MRNA therapy for pompe disease

ABSTRACT

The present invention provides, among other things, methods of treating Pompe disease, including administering to a subject in need of treatment a composition comprising an mRNA encoding acid alpha-glucosidase (GAA) at an effective dose and an administration interval such that at least one symptom or feature of Pompe disease is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/191,017, filed Nov. 14, 2018, which is issued as U.S. Pat. No.11,090,368 on Aug. 17, 2021, which is a continuation of U.S. applicationSer. No. 15/073,163, filed Mar. 17, 2016, which is issued as U.S. Pat.No. 10,172,924 on Jan. 8, 2019, which claims priority to U.S.Provisional Application Ser. No. 62/135,338, filed Mar. 19, 2015, thedisclosure of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named“SHR_1185US3_SequenceListing” on Jul. 7, 2021). The .txt file wasgenerated Jul. 7, 2021 and is 28,284 bytes in size. The entire contentsof the Sequence Listing are herein incorporated by reference.

BACKGROUND

Pompe disease (also known as glycogen storage disease type II; acidalpha-glucosidase deficiency; acid maltase deficiency; GAA deficiency;GSD II; glycogenosis type II; glycogenosis, generalized, cardiac form;cardiomegalia glycogenica diffusa; acid maltase deficiency; AMD; oralpha-1,4-glucosidase deficiency) is an autosomal recessive metabolicgenetic disorder characterized by mutations in the gene for thelysomsomal enzyme acid alpha-glucosidase (GAA) (also known as acidmaltase). Mutations in the GAA gene eliminate or reduce the ability ofthe GAA enzyme to hydrolyze the α-1,4 and α-1,6 linkages in glycogen,maltose and isomaltose. As a result, glycogen accumulates in thelysosomes and cytoplasm of cells throughout the body leading to cell andtissue destruction. Tissues that are particularly affected includeskeletal muscle and cardiac muscle. The accumulated glycogen causesprogressive muscle weakness leading to cardiomegaly, ambulatorydifficulties and respiratory insufficiency.

Three forms of Pompe disease have been identified, including the classicinfantile-onset disease, non-classic infantile-onset disease and lateonset disease. The classic infantile-onset form is characterized bymuscle weakness, poor muscle tone, hepatomegaly and cardiac defects. Theincidence of the disease is approximately 1 in 140,000 individuals.Patients with this form of the disease often die of heart failure in thefirst year of life. The non-classic infantile-onset form of the diseaseis characterized by delayed motor skills, progressive muscle weaknessand in some instances cardiomegaly. Patients with this form of thedisease often live only into early childhood due to respiratory failure.The late-onset form of the disease may present in late childhood,adolescence or adulthood and is characterized by progressive muscleweakness of the legs and trunk.

Currently, there is no cure for Pompe disease and the standard of careis enzyme replacement therapy (ERT) with supportive care forcardiomyopathy and physical therapy for muscle weakness and respiratorysymptoms.

SUMMARY OF THE INVENTION

The present invention provides, among other things, improved methods andcompositions for the treatment of Pompe disease based on mRNA therapy.The invention encompasses the observation that administration of an mRNAencoding a human GAA protein, encapsulated within a liposome, resultedin highly efficient and sustained protein production in vivo andsuccessful reduction of, for example, glycogen levels in the liver andmuscle, a clinically-relevant disease marker.

In one aspect, the present invention provides a method of treating Pompedisease, including administering to a subject in need of treatment acomposition comprising an mRNA encoding acid alpha-glucosidase (GAA) atan effective dose and an administration interval such that at least onesymptom or feature of Pompe disease is reduced in intensity, severity,or frequency or has delayed onset. In some embodiments, the mRNA isencapsulated within a liposome.

In another aspect, the present invention provides a method of treatingPompe disease, including administering to a subject in need of treatmenta therapeutically effective amount of a composition comprising an mRNAencoding acid alpha-glucosidase (GAA) such that hypertrophiccardiomyopathy in the subject is treated. In some embodiments, the mRNAis encapsulated within a liposome.

In another aspect, the present invention provides compositions fortreating Pompe disease comprising an mRNA encoding GAA at an effectivedose amount encapsulated within a liposome.

In some embodiments, a suitable liposome comprises one or more cationiclipids, one or more non-cationic lipids, one or more cholesterol-basedlipids and one or more PEG-modified lipids.

In some embodiments, the one or more cationic lipids are selected fromthe group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE(Imidazol-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS,DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA,DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA,HGT4003, and combinations thereof.

In some embodiments, the one or more cationic lipids comprise a compoundof formula I-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each R² independently is hydrogen or C₁₋₃ alkyl;    -   each q independently is 2 to 6;    -   each R′ independently is hydrogen or C₁₋₃ alkyl;    -   and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, the one or more cationic lipids comprise cKK-E12:

In some embodiments, the one or more non-cationic lipids suitable forthe invention are selected from DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and combinationsthereof.

In some embodiments, the one or more cholesterol-based lipids areselected from cholesterol, PEGylated cholesterol and DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine and combinations thereof.

In some embodiments, the liposome further comprises one or morePEG-modified lipids. In some embodiments, the one or more PEG-modifiedlipids comprise a poly(ethylene) glycol chain of up to 5 kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. Insome embodiments, a PEG-modified lipid is a derivatized ceramide such asN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000].In some embodiments, a PEG-modified or PEGylated lipid is PEGylatedcholesterol or Dimyristoylglycerol (DMG)-PEG-2K.

In some embodiments, a suitable liposome comprises a combinationselected from cKK-E12, DOPE, cholesterol and DMG-PEG2K; C12-200, DOPE,cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; orICE, DOPE, cholesterol and DMG-PEG2K.

In some embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE,and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%) of the liposome by molar ratio. In some embodiments, cationiclipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) constitute about30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%of the liposome by molar ratio.

In some embodiments, the ratio of cationic lipid(s) (e.g., cKK-E12,C12-200, ICE, and/or HGT4003) to non-cationic lipid(s) (e.g., DOPE) tocholesterol-based lipid(s) (e.g., cholesterol) to PEGylated lipid(s)(e.g., DMG-PEG2K) may be between about 30-60:25-35:20-30:1-15,respectively. In some embodiments, the ratio of cationic lipid(s) (e.g.,cKK-E12, C12-200, ICE, and/or HGT4003) to non-cationic lipid(s) (e.g.,DOPE) to cholesterol-based lipid(s) (e.g., cholesterol) to PEGylatedlipid(s) (e.g., DMG-PEG2K) is approximately 40:30:20:10, respectively.In some embodiments, the ratio of cationic lipid(s) (e.g., cKK-E12,C12-200, ICE, and/or HGT4003) to non-cationic lipid(s) (e.g., DOPE) tocholesterol-based lipid(s) (e.g., cholesterol) to PEGylated lipid(s)(e.g., DMG-PEG2K) is approximately 40:30:25:5, respectively. In someembodiments, the ratio of cationic lipid(s) (e.g., cKK-E12, C12-200,ICE, and/or HGT4003) to non-cationic lipid(s) (e.g., DOPE) tocholesterol-based lipid(s) (e.g., cholesterol) to PEGylated lipid(s)(e.g., DMG-PEG2K) is approximately 40:32:25:3, respectively. In someembodiments, the ratio of cationic lipid(s) (e.g., cKK-E12, C12-200,ICE, and/or HGT4003) to non-cationic lipid(s) (e.g., DOPE) tocholesterol-based lipid(s) (e.g., cholesterol) to PEGylated lipid(s)(e.g., DMG-PEG2K) is approximately 50:25:20:5.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the liposome particle. In some embodiments, asuitable liposome has a size less than about 500 nm, 400 nm, 300 nm, 250nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm. In some embodiments, asuitable liposome has a size less than about 100 nm, 90 nm, 80 nm, 70nm, or 60 nm. In a particular embodiment, the liposome has a size lessthan about 100 nm.

In some embodiments, the mRNA is administered at a dose ranging fromabout 0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0,0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3,0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5,0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5,0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, themRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5,3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg bodyweight. In a particular embodiment, the mRNA is administered at a doseof about 1.0 mg/kg.

In some embodiments, the provided composition is administeredintravenously. In some embodiments, the provided composition isadministered intramuscularly. In particular embodiments, theintramuscular administration is to a muscle selected from the groupconsisting of skeletal muscle, smooth muscle and cardiac muscle. In someembodiments, the provided composition is administered via pulmonarydelivery. In certain embodiments, pulmonary delivery is performed byaerosolization, inhalation, nebulization or instillation. In someembodiments, the provided composition is formulated as respirableparticles, nebulizable lipid, or inhalable dry powder.

In some embodiments, the provided composition is administered oncedaily, once a week, twice a week, twice a month, once a month. In someembodiments, provided the composition is administered once every 7 days,once every 10 days, once every 14 days, once every 28 days or once every30 days.

In some embodiments, a therapeutically effective dose, when administeredregularly, results in GAA protein expression in the liver. In someembodiments, a therapeutically effective dose, when administeredregularly, results in GAA protein expression in a muscle tissue or amuscle cell. The muscle tissue may be, for example, skeletal muscle,smooth muscle, cardiac muscle and combinations thereof. The muscle cellmay be, for example, a myocyte, a myotube, a myoblast, a cardiomyocyte,a cardiomyoblast and combinations thereof. In some embodiments, atherapeutically effective dose, when administered regularly, results inGAA protein detection in serum.

In some embodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in an increasedhepatic GAA protein level in a subject as compared to a baseline hepaticGAA protein level before treatment. In some embodiments, atherapeutically effective dose of the provided composition, whenadministered regularly, results in an increased muscle GAA protein levelin a subject as compared to a baseline muscle GAA protein level beforetreatment. In some embodiments, the muscle is skeletal muscle (e.g.,striated muscle, voluntary muscle), smooth muscle (e.g., visceralmuscle, involuntary muscle) or cardiac muscle. In some embodiments, atherapeutically effective dose of the provided composition, whenadministered regularly, results in a reduced muscle glycogen level in asubject as compared to a baseline muscle glycogen level beforetreatment. In some embodiments, a therapeutically effective dose of theprovided composition, when administered regularly, results in a reducedliver glycogen level in a subject as compared to a baseline liverglycogen level before treatment. In some embodiments, a therapeuticallyeffective dose of the provided composition, when administered regularly,results in a reduced serum creatine kinase level in a subject ascompared to a baseline serum creatine kinase level before treatment. Insome embodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in a reduced urinaryglucose tetrasaccharide, (Glcα1-6Glcα1-4Glcα1-4Glc (Glc₄) level in asubject as compared to a baseline Glc₄ level before treatment. In someembodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in a reduced serumaspartate transaminase (e.g., AST, aspartate aminotransferase, serum,glutamic oxaloacetic transaminase) level in a subject as compared to abaseline AST level before treatment. In some embodiments, atherapeutically effective dose of the provided composition, whenadministered regularly, results in a reduced serum alanine transaminase(e.g., ALT, alanine aminotransferase, serum glutamic-pyruvictransaminase) level in a subject as compared to a baseline ALT levelbefore treatment. In some embodiments, a therapeutically effective doseof the provided composition, when administered regularly, results in areduced serum lactate dehydrogenase (e.g., LDH, lactic dehydrogenase)level in a subject as compared to a baseline LDH level before treatment.In some embodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in an increased GAAenzyme activity level in biological sample as compared to a baseline GAAenzyme activity level before treatment.

In some embodiments, administering the provided composition results inan increased GAA protein level in the liver of a subject as compared toa baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in the liver by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in the liver as compared to aGAA protein level in the liver of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased GAA protein level in skeletal muscle of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in skeletal muscle by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in skeletal muscle as comparedto a GAA protein level in skeletal muscle of subjects who are nottreated.

In some embodiments, administering the provided composition results inan increased GAA protein level in cardiac muscle of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in cardiac muscle by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in cardiac muscle as comparedto a GAA protein level in cardiac muscle of subjects who are nottreated.

In some embodiments, administering the provided composition results inan increased level of GAA protein in smooth muscle of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in smooth muscle by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in smooth muscle as comparedto a GAA protein level in smooth muscle of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased level of GAA protein in a muscle cell of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments themuscle cell is a myocyte, a myotube, a myoblast, a cardiomyocyte or acardiomyoblast. In some embodiments, administering the providedcomposition results in an increased GAA protein level in the muscle cellby at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in a muscle cell as compared to a GAA protein level in amuscle cell of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased level of GAA protein in a liver cell (e.g., a hepatocyte, asinusoid lining cell) of a subject as compared to a baseline levelbefore treatment. Typically, the baseline level is measured immediatelybefore treatment. In some embodiments, administering the providedcomposition results in an increased GAA protein level in the liver cellby at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in a liver cell as compared to a GAA protein level in aliver cell of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased GAA protein level in plasma or serum of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in plasma or serum by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in plasma or serum as comparedto a GAA protein level in plasma or serum of subjects who are nottreated

In some embodiments, administering the provided composition results in areduced serum creatine kinase level in a subject as compared to abaseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serumcreatine kinase level by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% as compared to the baseline serum creatine kinaselevel immediately before treatment. In some embodiments, administeringthe provided composition results in a reduced serum creatine kinaselevel to less than about 2000 IU/L, 1500 IU/L, 1000 IU/L, 750 IU/L, 500IU/L, 250 IU/L, 100 IU/L, 90 IU/L, 80 IU/L, 70 IU/L or 60 IU/L. In someembodiments, administering the provided composition results in a reducedserum creatine kinase level as compared to a serum creatine kinase levelin subjects who are not treated.

In some embodiments, administering the provided composition results in areduced urinary Glc₄ level in a subject as compared to a baseline levelbefore treatment. Typically, the baseline level is measured immediatelybefore treatment. In some embodiments, administering the providedcomposition results in a reduced urinary Glc₄ level by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to abaseline level immediately before treatment. In some embodiments,administering the provided composition results in a reduced urinary Glc₄level to less than about 100 mmol Glc₄/mol creatinine, 90 mmol Glc₄/molcreatinine, 80 mmol Glc₄/mol creatinine, 70 mmol Glc₄/mol creatinine, 60mmol Glc₄/mol creatinine, 50 mmol Glc₄/mol creatinine, 40 mmol Glc₄/molcreatinine, 30 mmol Glc₄/mol creatinine or 20 mmol Glc₄/mol creatinine.In some embodiments, administering the provided composition results in areduced urinary Glc₄ level as compared to a urinary Glc₄ level insubjects who are not treated.

In some embodiments, administering the provided composition results in areduced muscle glycogen level in a subject as compared to a baselinelevel before treatment. Typically, the baseline level is measuredimmediately before treatment. In some embodiments, administering theprovided composition results in a reduced muscle glycogen level by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level immediately before treatment. In someembodiments, administering the provided composition results in a reducedmuscle glycogen level as compared to a muscle glycogen level in subjectswho are not treated. In particular embodiments, the muscle is skeletalmuscle, smooth muscle or cardiac muscle.

In some embodiments, administering the provided composition results in areduced liver glycogen level in a subject as compared to a baselinelevel before treatment. Typically, the baseline level is measuredimmediately before treatment. In some embodiments, administering theprovided composition results in a reduced liver glycogen level by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level immediately before treatment. In someembodiments, administering the provided composition results in a reducedliver glycogen level as compared to a liver glycogen level in subjectswho are not treated.

In some embodiments, administering the provided composition results in areduced serum aspartate transaminase (AST) level in a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serum ASTlevel by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or95% as compared to a baseline level immediately before treatment. Insome embodiments, administering the provided composition results in areduced serum AST level to less than about 600 IU/L, 500 IU/L, 400 IU/L,300 IU/L, 200 IU/L, 100 IU/L, 50 IU/L, 25 IU/L, 20 IU/L or 10 IU/L. Insome embodiments, administering the provided composition results in areduced serum AST level as compared to a serum AST level in subjects whoare not treated.

In some embodiments, administering the provided composition results in areduced serum alanine transaminase (ALT) level in a subject as comparedto a baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serum ALTlevel by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or95% as compared to a baseline level immediately before treatment. Insome embodiments, administering the provided composition results in areduced serum ALT level to less than about 1000 IU/L, 900 IU/L, 800IU/L, 700 IU/L, 600 IU/L, 500 IU/L, 400 IU/L, 300 IU/L, 200 IU/L, 100IU/L, 50 IU/L, 25 IU/L, 20 IU/L or 10 IU/L. In some embodiments,administering the provided composition results in a reduced serum ALTlevel as compared to a serum ALT level in subjects who are not treated.

In some embodiments, administering the provided composition results in areduced serum lactate dehydrogenase (LDH) level in a subject as comparedto a baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serumlactate dehydrogenase LDH level by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline levelimmediately before treatment. In some embodiments, administering theprovided composition results in a reduced serum LDH level to less thanabout 2000 IU/L, 1500 IU/L, 1000 IU/L, 900 IU/L, 800 IU/L, 700 IU/L, 600IU/L, 500 IU/L, 400 IU/L, 300 IU/L, 200 IU/L or 100 IU/L. In someembodiments, administering the provided composition results in a reducedserum LDH level as compared to a serum LDH levels in subjects who arenot treated.

In some embodiments, administering the provided composition results inan increased GAA enzyme activity in a biological sample from a subjectas compared to a baseline level before treatment. Typically, thebaseline level is measured immediately before treatment. Biologicalsamples include, for example, whole blood, serum, plasma, urine andtissue samples (e.g., muscle, liver, skin fibroblasts). In someembodiments, administering the provided composition results in anincreased GAA enzyme activity by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% as compared to a baseline level immediatelybefore treatment. In some embodiments, administering the providedcomposition results in an increased GAA enzyme activity as compared to aGAA enzyme activity in subjects who are not treated.

In some embodiments, administering the provided composition results inan increased GAA mRNA expression level in a biological sample from asubject as compared to a baseline expression level before treatment.Typically, the baseline level is measured immediately before treatment.Biological samples include, for example, whole blood, serum, plasma,urine and tissue samples (e.g., muscle, liver, skin fibroblasts). Insome embodiments, administering the provided composition results in anincreased GAA mRNA expression level by at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline levelimmediately before treatment. In some embodiments, administering theprovided composition results in an increased GAA mRNA expression levelas compared to a GAA mRNA expression level in subjects who are nottreated.

In some embodiments, the mRNA is codon optimized. In some embodiments,the codon-optimized mRNA comprises SEQ ID NO: 3 (corresponding tocodon-optimized human GAA mRNA sequences). In some embodiments, the mRNAcomprises the 5′ UTR sequence of SEQ ID NO: 8 (corresponding to 5′ UTRsequence X). In some embodiments, the mRNA comprises the 3′ UTR sequenceof SEQ ID NO: 9 (corresponding to a 3′ UTR sequence Y). In someembodiments, the mRNA comprises the 3′ UTR sequence of SEQ ID NO: 10(corresponding to a 3′ UTR sequence Y). In some embodiments, thecodon-optimized mRNA comprises SEQ ID NO: 11 or SEQ ID NO: 12(corresponding to codon-optimized human GAA mRNA sequence with 5′ UTRand 3′ UTR sequences).

In some embodiments, the mRNA comprises one or more modifiednucleotides. In some embodiments, the one or more modified nucleotidescomprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, 0(6)-methylguanine, and/or 2-thiocytidine. In someembodiments, the mRNA is unmodified.

In one aspect, the present invention provides a composition for treatingPompe disease, comprising an mRNA encoding acid alpha-glucosidase (GAA)at an effective dose amount encapsulated within a liposome, wherein theliposome comprises a cationic lipid cKK-E12:

In a one embodiment, the liposome further comprises one or morenon-cationic lipids, one or more cholesterol-based lipids, and one ormore PEG-modified lipids. In some embodiments, the one or morenon-cationic lipids are selected from DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).

In another embodiment, the one or more cholesterol-based lipids areselected from cholesterol and/or PEGylated cholesterol. In a furtherembodiment, the one or more PEG-modified lipids comprise apoly(ethylene) glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C₆-C₂₀ length.

In another embodiment, the liposome comprises cKK-E12, DOPE, cholesteroland DMG-PEG2K. In a particular embodiment, the cationic lipidconstitutes about 30-50% of the liposome by molar ratio. In anotherembodiment, the cationic lipid constitutes about 40% of the liposome bymolar ratio.

In another embodiment, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2Kis approximately 40:30:20:10 by molar ratio. In a particular embodiment,the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately40:30:25:5 by molar ratio. In yet another embodiment, the ratio ofcKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molarratio. In one embodiment, the liposome has a size less than about 100nm.

In one embodiment, the composition is formulated for intravenousadministration. In another embodiment, the composition is formulated forintramuscular administration. In another embodiment, the mRNA comprisesSEQ ID NO: 3. In a further embodiment, the mRNA further comprises the 5′UTR sequence of SEQ ID NO: 8. In yet another embodiment, the mRNAfurther comprises the 3′ UTR sequence of SEQ ID NO: 9 or SEQ ID NO: 10.In a particular embodiment, the mRNA comprises SEQ ID NO: 11 or SEQ IDNO: 12.

In one aspect, the present invention provides a composition for treatingPompe disease comprising an mRNA encoding acid alpha-glucosidase (GAA)at an effective dose amount encapsulated within a liposome, wherein themRNA comprises SEQ ID NO: 3, and further wherein the liposome comprisescationic or non-cationic lipid, cholesterol-based lipid and PEG-modifiedlipid.

In in one aspect, the present invention provides a composition fortreating Pompe disease comprising an mRNA encoding acidalpha-glucosidase (GAA) at an effective dose amount encapsulated withina liposome, wherein the mRNA comprises SEQ ID NO: 11 or SEQ ID NO: 12,and further wherein the liposome comprises cationic or non-cationiclipid, cholesterol-based lipid and PEG-modified lipid.

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The drawings are for illustration purposes only not for limitation.

FIGS. 1A-1B depict exemplary GAA mRNA detection by in situ hybridizationin muscle tissue from mice 6 hours (FIG. 1A) or 12 hours (FIG. 1B) aftertreatment with a single 1.0 mg/kg intravenous dose of GAA mRNAencapsulated lipid nanoparticles.

FIG. 2A depicts exemplary glycogen reduction in livers of GAA knock-outmice 24 hours after treatment with a single 1.0 mg/kg intravenous doseof GAA mRNA encapsulated lipid nanoparticles.

FIG. 2B depicts exemplary glycogen accumulation in livers of GAAknock-out mice that were not treated with GAA mRNA encapsulated lipidnanoparticles.

FIG. 3 depicts exemplary GAA mRNA detection by in situ hybridization inmuscle tissue from mice 24 hours after treatment with a single 1.0 mg/kgintramuscular dose of GAA mRNA encapsulated lipid nanoparticles.

FIGS. 4A-4B depict exemplary glycogen reduction in quadriceps muscle ofGAA knock-out mice 24 hours after treatment with a single 1.0 mg/kgintramuscular dose of GAA mRNA encapsulated lipid nanoparticles.

FIGS. 4C-4D depict exemplary glycogen levels in quadriceps muscle of GAAknock-out mice that were not treated with GAA mRNA encapsulated lipidnanoparticles.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

Alkyl: As used herein, “alkyl” refers to a radical of a straight-chainor branched saturated hydrocarbon group having from 1 to 15 carbon atoms(“C₁₋₁₅ alkyl”). In some embodiments, an alkyl group has 1 to 3 carbonatoms (“C₁₋₃ alkyl”). Examples of C₁₋₃ alkyl groups include methyl (C₁),ethyl (C₂), n-propyl (C₃), and isopropyl (C₃). In some embodiments, analkyl group has 8 to 12 carbon atoms (“C₈₋₁₂ alkyl”). Examples of C₈₋₁₂alkyl groups include, without limitation, n-octyl (C₈), n-nonyl (C₉),n-decyl (C₁₀), n-undecyl (C₁₁), n-dodecyl (Cu) and the like. The prefix“n-” (normal) refers to unbranched alkyl groups. For example, n-C₈ alkylrefers to —(CH₂)₇CH₃, n-C₁₀ alkyl refers to —(CH₂)₉CH₃, etc.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active.

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinis expressed and retained within the target tissue (also referred to as“local distribution” or “local delivery”), and situations in which anmRNA is delivered to a target tissue and the encoded protein isexpressed and secreted into patient's circulation system (e.g., serum)and systematically distributed and taken up by other tissues (alsoreferred to as “systemic distribution” or “systemic delivery).

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a polypeptide, assemble multiplepolypeptides into an intact protein (e.g., enzyme) and/orpost-translational modification of a polypeptide or fully assembledprotein (e.g., enzyme). In this application, the terms “expression” and“production,” and grammatical equivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required fora quantity such as nucleic acid or protein concentration or activity tofall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “localdistribution,” “local delivery,” or grammatical equivalent, refer totissue specific delivery or distribution. Typically, local distributionor delivery requires a protein (e.g., enzyme) encoded by mRNAs betranslated and expressed intracellularly or with limited secretion thatavoids entering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide. mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions. mRNA can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, mRNA can comprisenucleoside analogs such as analogs having chemically modified bases orsugars, backbone modifications, etc. An mRNA sequence is presented inthe 5′ to 3′ direction unless otherwise indicated. In some embodiments,an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine,cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemicallymodified bases; biologically modified bases (e.g., methylated bases);intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

Muscle cell or muscle tissue: As used herein, the term “muscle cell” or“muscle tissue” in its broadest sense, refers to a cell or group ofcells derived from muscle, including, but not limited to cells andtissues derived from skeletal muscle (e.g., striated muscle, voluntarymuscle); smooth muscle (e.g., visceral muscle, involuntary muscle) fromthe digestive tract, urinary bladder and blood vessels; and cardiacmuscle. The term refers to muscle cells in vivo and in vitro. The termalso includes differentiated and undifferentiated, or nondifferentiated,muscle cells, such as myocytes, myotubes, myoblasts, cardiomyocytes andcardiomyoblasts.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable salt: Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge et al., describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or rnalonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium. quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequarternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

Systemic distribution or delivery: As used herein, the terms “systemicdistribution,” “systemic delivery,” or grammatical equivalent, refer toa delivery or distribution mechanism or approach that affect the entirebody or an entire organism. Typically, systemic distribution or deliveryis accomplished via body's circulation system, e.g., blood stream.Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, methods andcompositions for treating Pompe disease based on mRNA therapy. Inparticular, the present invention provides methods for treating Pompedisease by administering to a subject in need of treatment a compositioncomprising an mRNA encoding acid alpha-glucosidase (GAA) at an effectivedose and an administration interval such that at least one symptom orfeature of Pome disease is reduced in intensity, severity, or frequencyor has a delayed onset. The present invention further provides, methodsof treating Pompe disease, comprising administering to a subject in needof treatment a therapeutically effective amount of a compositioncomprising an mRNA encoding acid alpha-glucosidase (GAA) such thathypertrophic cardiomyopathy in the subject is treated. In someembodiments, the mRNA is encapsulated within one or more liposomes. Asused herein, the term “liposome” refers to any lamellar, multilamellar,or solid nanoparticle vesicle. Typically, a liposome as used herein canbe formed by mixing one or more lipids or by mixing one or more lipidsand polymer(s). Thus, the term “liposome” as used herein encompassesboth lipid and polymer based nanoparticles. In some embodiments, aliposome suitable for the present invention contains cationic ornon-cationic lipid(s), cholesterol-based lipid(s) and PEG-modifiedlipid(s).

Pompe Disease

The present invention may be used to treat a subject who is sufferingfrom or susceptible to Pompe disease. Pompe disease is an autosomalrecessive metabolic genetic disorder characterized by mutations in thegene for the enzyme acid alpha-glucosidase (GAA). The GAA enzyme is alsoknown as: acid maltase, maltase, maltase-glucoamylase, gluoinvertase,glucosidosucrase, aglucosidase alfa, alpha-1,4-glucosidase,amyloglucosidase, glucoamylase, LYAG, LYAG_HUMAN, lysosomalalpha-glucosidase and alpha-glucosidase, acid. The GAA gene(glucosidase, alpha; acid) is also known as: acid maltase,alpha-1,4-glucosidase and alpha-gluosidase, acid. More than 200mutations that cause Pompe disease have been identified in the GAA gene.Most of these mutations involve single amino acid substitutions andsmall insertions or deletions. Many of the mutations in the GAA genelikely affect the structure of the resulting protein and decrease itsactivity. A few of the mutations in the GAA gene lead to the productionof an abnormally short version of the enzyme that cannot effectivelyplay its role in the hydrolysis of glycogen.

Defects in the acid alpha-glucosidase enzyme reduce the ability of thecell to hydrolyze glycogen resulting in the accumulation of glycogen incells and tissues, and in particular in the liver and muscle. Theaccumulation of glycogen results in cell death which manifests asprogressive muscle weakness, cardiomyopathy and respiratoryinsufficiency.

Compositions and methods described herein may be used to treat at leastone symptom or feature of Pompe disease. In particular, the compositionsand methods described herein may be used to treat hypertrophiccardiomyopathy.

Acid Alpha-Glucosidase (GAA)

In some embodiments, the present invention provides methods andcompositions for delivering mRNA encoding GAA to a subject for thetreatment of Pompe disease. A suitable GAA mRNA encodes any full length,fragment or portion of an GAA protein which can be substituted fornaturally-occurring GAA protein activity and/or reduce the intensity,severity, and/or frequency of one or more symptoms associated with Pompedisease.

In some embodiments, a suitable mRNA sequence is an mRNA sequenceencoding a human GAA protein. The naturally-occurring human GAA mRNAcoding sequence and the corresponding amino acid sequence are shown inTable 1:

TABLE 1 Human GAA Human (SEQ ID NO: 1) GAAAUGGGAGUGAGGCACCCGCCCUGCUCCCACCGGC (mRNAUCCUGGCCGUCUGCGCCCUCGUGUCCUUGGCAAC codingCGCUGCACUCCUGGGGCACAUCCUACUCCAUGAU sequence)UUCCUGCUGGUUCCCCGAGAGCUGAGUGGCUCCU CCCCAGUCCUGGAGGAGACUCACCCAGCUCACCAGCAGGGAGCCAGCAGACCAGGGCCCCGGGAUGCC CAGGCACACCCCGGCCGUCCCAGAGCAGUGCCCACACAGUGCGACGUCCCCCCCAACAGCCGCUUCGA UUGCGCCCCUGACAAGGCCAUCACCCAGGAACAGUGCGAGGCCCGCGGCUGUUGCUACAUCCCUGCAA AGCAGGGGCUGCAGGGAGCCCAGAUGGGGCAGCCCUGGUGCUUCUUCCCACCCAGCUACCCCAGCUAC AAGCUGGAGAACCUGAGCUCCUCUGAAAUGGGCUACACGGCCACCCUGACCCGUACCACCCCCACCUU CUUCCCCAAGGACAUCCUGACCCUGCGGCUGGACGUGAUGAUGGAGACUGAGAACCGCCUCCACUUCA CGAUCAAAGAUCCAGCUAACAGGCGCUACGAGGUGCCCUUGGAGACCCCGCAUGUCCACAGCCGGGCA CCGUCCCCACUCUACAGCGUGGAGUUCUCCGAGGAGCCCUUCGGGGUGAUCGUGCGCCGGCAGCUGGA CGGCCGCGUGCUGCUGAACACGACGGUGGCGCCCCUGUUCUUUGCGGACCAGUUCCUUCAGCUGUCCA CCUCGCUGCCCUCGCAGUAUAUCACAGGCCUCGCCGAGCACCUCAGUCCCCUGAUGCUCAGCACCAGC UGGACCAGGAUCACCCUGUGGAACCGGGACCUUGCGCCCACGCCCGGUGCGAACCUCUACGGGUCUCA CCCUUUCUACCUGGCGCUGGAGGACGGCGGGUCGGCACACGGGGUGUUCCUGCUAAACAGCAAUGCCA UGGAUGUGGUCCUGCAGCCGAGCCCUGCCCUUAGCUGGAGGUCGACAGGUGGGAUCCUGGAUGUCUAC AUCUUCCUGGGCCCAGAGCCCAAGAGCGUGGUGCAGCAGUACCUGGACGUUGUGGGAUACCCGUUCAU GCCGCCAUACUGGGGCCUGGGCUUCCACCUGUGCCGCUGGGGCUACUCCUCCACCGCUAUCACCCGCC AGGUGGUGGAGAACAUGACCAGGGCCCACUUCCCCCUGGACGUCCAGUGGAACGACCUGGACUACAUG GACUCCCGGAGGGACUUCACGUUCAACAAGGAUGGCUUCCGGGACUUCCCGGCCAUGGUGCAGGAGCU GCACCAGGGCGGCCGGCGCUACAUGAUGAUCGUGGAUCCUGCCAUCAGCAGCUCGGGCCCUGCCGGGA GCUACAGGCCCUACGACGAGGGUCUGCGGAGGGGGGUUUUCAUCACCAACGAGACCGGCCAGCCGCUG AUUGGGAAGGUAUGGCCCGGGUCCACUGCCUUCCCCGACUUCACCAACCCCACAGCCCUGGCCUGGUG GGAGGACAUGGUGGCUGAGUUCCAUGACCAGGUGCCCUUCGACGGCAUGUGGAUUGACAUGAACGAGC CUUCCAACUUCAUCAGGGGCUCUGAGGACGGCUGCCCCAACAAUGAGCUGGAGAACCCACCCUACGUG CCUGGGGUGGUUGGGGGGACCCUCCAGGCGGCCACCAUCUGUGCCUCCAGCCACCAGUUUCUCUCCAC ACACUACAACCUGCACAACCUCUACGGCCUGACCGAAGCCAUCGCCUCCCACAGGGCGCUGGUGAAGG CUCGGGGGACACGCCCAUUUGUGAUCUCCCGCUCGACCUUUGCUGGCCACGGCCGAUACGCCGGCCAC UGGACGGGGGACGUGUGGAGCUCCUGGGAGCAGCUCGCCUCCUCCGUGCCAGAAAUCCUGCAGUUUAA CCUGCUGGGGGUGCCUCUGGUCGGGGCCGACGUCUGCGGCUUCCUGGGCAACACCUCAGAGGAGCUGU GUGUGCGCUGGACCCAGCUGGGGGCCUUCUACCCCUUCAUGCGGAACCACAACAGCCUGCUCAGUCUG CCCCAGGAGCCGUACAGCUUCAGCGAGCCGGCCCAGCAGGCCAUGAGGAAGGCCCUCACCCUGCGCUA CGCACUCCUCCCCCACCUCUACACACUGUUCCACCAGGCCCACGUCGCGGGGGAGACCGUGGCCCGGC CCCUCUUCCUGGAGUUCCCCAAGGACUCUAGCACCUGGACUGUGGACCACCAGCUCCUGUGGGGGGAG GCCCUGCUCAUCACCCCAGUGCUCCAGGCCGGGAAGGCCGAAGUGACUGGCUACUUCCCCUUGGGCAC AUGGUACGACCUGCAGACGGUGCCAGUAGAGGCCCUUGGCAGCCUCCCACCCCCACCUGCAGCUCCCC GUGAGCCAGCCAUCCACAGCGAGGGGCAGUGGGUGACGCUGCCGGCCCCCCUGGACACCAUCAACGUC CACCUCCGGGCUGGGUACAUCAUCCCCCUGCAGGGCCCUGGCCUCACAACCACAGAGUCCCGCCAGCA GCCCAUGGCCCUGGCUGUGGCCCUGACCAAGGGUGGGGAGGCCCGAGGGGAGCUGUUCUGGGACGAUG GAGAGAGCCUGGAAGUGCUGGAGCGAGGGGCCUACACACAGGUCAUCUUCCUGGCCAGGAAUAACACG AUCGUGAAUGAGCUGGUACGUGUGACCAGUGAGGGAGCUGGCCUGCAGCUGCAGAAGGUGACUGUCCU GGGCGUGGCCACGGCGCCCCAGCAGGUCCUCUCCAACGGUGUCCCUGUCUCCAACUUCACCUACAGCC CCGACACCAAGGUCCUGGACAUCUGUGUCUCGCUGUUGAUGGGAGAGCAGUUUCUCGUCAGCUGGUGU UAG Human (SEQ ID NO: 2) GAAMGVRHPPCSHRLLAVCALVSLATAALLGHILLHD (AminoFLLVPRELSGSSPVLEETHPAHQQGASRPGPRDA AcidQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQ Sequence)CEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSY KLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPHVHSRA PSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTS WTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVY IFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYM DSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPL IGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYV PGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGH WTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSL PQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGE ALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINV HLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNT IVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC

In some embodiments, a suitable mRNA is a wild-type human GAA mRNA ofsequence (SEQ ID NO: 1). In some embodiments, a suitable mRNA may be acodon optimized hGAA sequence, such as the sequence shown below:

Codon-Optimized human GAA Coding Sequence (SEQ ID NO: 3):AUGGGAGUCAGACACCCGCCGUGCUCGCACAGGCUUCUGGCCGUGUGCGCACUCGUGAGUCUGGCGACUGCUGCGUUGCUGGGGCACAUUCUUCUCCACGACUUUCUCUUGGUGCCCCGAGAAUUGUCGGGCUCGUCGCCGGUACUGGAAGAAACCCACCCCGCACAUCAGCAGGGCGCGUCGCGGCCUGGUCCGAGGGAUGCCCAGGCACAUCCCGGAAGGCCACGAGCCGUCCCGACUCAAUGUGACGUACCUCCCAAUUCCCGGUUCGACUGUGCGCCAGACAAGGCAAUCACGCAAGAGCAGUGCGAAGCCCGUGGAUGCUGCUAUAUUCCGGCGAAGCAGGGACUUCAGGGAGCCCAGAUGGGGCAGCCCUGGUGUUUCUUCCCGCCUUCCUAUCCCUCAUAUAAGCUGGAGAAUUUGUCGUCCUCGGAAAUGGGGUAUACCGCUACUCUUACGAGAACCACCCCCACAUUCUUUCCGAAGGACAUCCUUACUCUGCGGCUCGACGUGAUGAUGGAGACAGAAAAUAGGCUGCAUUUCACGAUCAAAGACCCGGCGAACCGGAGAUAUGAGGUUCCGCUUGAGACUCCCCACGUUCACUCUCGUGCGCCUUCACCCUUGUACUCCGUGGAGUUCUCGGAAGAACCGUUCGGGGUGAUCGUCAGACGUCAACUUGAUGGUAGGGUAUUGCUGAACACAACGGUCGCCCCCUUGUUUUUCGCCGACCAGUUUCUGCAGCUUUCGACAUCGCUGCCGUCCCAGUAUAUCACAGGGCUCGCGGAGCAUCUUUCACCCCUCAUGCUGAGCACGAGCUGGACACGGAUUACGCUCUGGAACAGGGAUCUCGCGCCGACGCCCGGAGCGAAUUUGUAUGGGUCGCAUCCCUUCUACCUCGCAUUGGAAGACGGGGGUUCCGCGCACGGAGUAUUCCUGCUUAAUUCUAAUGCGAUGGACGUUGUCUUGCAGCCCUCCCCUGCUUUGUCGUGGCGUUCCACGGGGGGCAUUUUGGACGUUUACAUCUUUUUGGGACCCGAGCCAAAGAGCGUAGUGCAGCAGUAUUUGGAUGUAGUGGGCUACCCCUUCAUGCCGCCUUAUUGGGGACUGGGGUUCCAUCUCUGCCGCUGGGGGUACUCUUCGACCGCGAUCACCCGCCAGGUGGUCGAGAACAUGACCAGAGCACAUUUCCCUUUGGACGUGCAGUGGAAUGAUUUGGAUUACAUGGAUAGCCGAAGAGACUUCACGUUCAAUAAGGACGGGUUUAGAGAUUUUCCCGCGAUGGUGCAAGAAUUGCACCAGGGUGGGCGCAGAUACAUGAUGAUCGUCGAUCCCGCCAUCAGCAGCUCGGGACCAGCGGGGAGUUACCGGCCUUACGAUGAGGGACUUAGGAGAGGCGUCUUUAUCACGAACGAAACAGGUCAGCCGCUCAUUGGUAAAGUGUGGCCUGGAUCAACGGCCUUUCCCGACUUCACGAAUCCCACAGCCCUCGCCUGGUGGGAAGACAUGGUGGCGGAGUUUCACGACCAAGUACCGUUUGAUGGGAUGUGGAUUGAUAUGAACGAACCCUCAAACUUUAUUCGCGGCUCGGAAGAUGGAUGCCCGAAUAAUGAGCUUGAGAAUCCCCCGUAUGUGCCAGGGGUGGUAGGUGGGACGCUCCAGGCCGCUACGAUCUGUGCGUCAUCACAUCAGUUCUUGUCAACGCACUACAACUUGCACAAUCUUUACGGUUUGACUGAAGCCAUCGCUUCGCAUCGCGCGCUGGUCAAAGCGCGUGGUACGCGACCCUUCGUUAUUUCUCGGUCCACAUUUGCCGGGCACGGUCGGUAUGCCGGACACUGGACGGGAGAUGUCUGGUCUAGCUGGGAGCAGCUCGCGUCGAGCGUACCGGAGAUCCUCCAGUUCAAUCUUUUGGGAGUUCCGCUCGUCGGCGCUGACGUGUGCGGUUUUCUCGGAAACACAUCAGAAGAGCUUUGCGUACGCUGGACACAGCUCGGUGCGUUUUACCCCUUUAUGAGAAACCAUAACUCGUUGCUCUCACUCCCUCAAGAGCCGUACAGUUUUUCGGAGCCUGCGCAACAGGCGAUGCGGAAGGCAUUGACACUUCGCUAUGCACUGCUCCCGCAUCUCUAUACUCUGUUCCAUCAGGCCCAUGUGGCUGGAGAAACGGUGGCGAGGCCCCUGUUCUUGGAGUUCCCCAAAGAUAGUUCCACAUGGACCGUGGAUCACCAGUUGCUGUGGGGAGAGGCGCUUCUGAUCACUCCGGUACUUCAGGCGGGUAAAGCGGAAGUCACUGGGUAUUUCCCGCUUGGGACCUGGUACGACCUUCAGACUGUCCCAGUAGAAGCCCUCGGAAGCCUGCCACCUCCCCCUGCUGCACCCCGCGAGCCUGCAAUCCAUAGCGAGGGCCAGUGGGUAACGUUGCCAGCCCCACUGGAUACCAUCAAUGUCCACCUCAGGGCGGGUUACAUUAUCCCUCUCCAAGGCCCUGGGUUGACCACCACAGAGUCGCGCCAGCAGCCAAUGGCACUUGCGGUCGCAUUGACGAAAGGGGGUGAAGCCCGAGGGGAACUGUUUUGGGAUGACGGGGAAAGCCUUGAGGUGCUGGAACGGGGAGCGUACACACAAGUCAUUUUCUUGGCCAGGAACAACACUAUUGUCAACGAGUUGGUGCGCGUGACCUCUGAGGGUGCCGGACUGCAACUGCAGAAGGUCACGGUCCUCGGAGUGGCGACAGCACCCCAACAGGUCCUUAGUAACGGAGUACCUGUCUCGAACUUUACAUACUCCCCGGACACGAAGGUGCUCGACAUCUGUGUGUCGCUGCUUAUGGGGGAACAGUUUC UCGUGAGCUGGUGCUAG

Additional exemplary mRNA sequences are described in the Examplessection below, for example, SEQ ID NO: 11 and SEQ ID NO: 12, both ofwhich include 5′ and 3′ untranslated regions framing a codon-optimizedGAA-encoding mRNA.

In some embodiments, a suitable mRNA sequence may be an mRNA sequence ahomolog or an analog of human GAA protein. For example, a homologue oran analogue of human GAA protein may be a modified human GAA proteincontaining one or more amino acid substitutions, deletions, and/orinsertions as compared to a wild-type or naturally-occurring human GAAprotein while retaining substantial GAA protein activity. In someembodiments, an mRNA suitable for the present invention encodes an aminoacid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:2. In some embodiments, an mRNA suitable for the present inventionencodes a protein substantially identical to human GAA protein. In someembodiments, an mRNA suitable for the present invention encodes an aminoacid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. In some embodiments, an mRNA suitable for the present inventionencodes a fragment or a portion of human GAA protein. In someembodiments, an mRNA suitable for the present invention encodes afragment or a portion of human GAA protein, wherein the fragment orportion of the protein still maintains GAA activity similar to that ofthe wild-type protein. In some embodiments, an mRNA suitable for thepresent invention has a nucleotide sequence at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11 or SEQ IDNO: 12.

In some embodiments, a suitable mRNA encodes a fusion protein comprisinga full length, fragment or portion of an GAA protein fused to anotherprotein (e.g., an N or C terminal fusion). In some embodiments, theprotein fused to the mRNA encoding a full length, fragment or portion ofan GAA protein encodes a signal or a cellular targeting sequence.

Delivery Vehicles

According to the present invention, mRNA encoding an GAA protein (e.g.,a full length, fragment or portion of an GAA protein) as describedherein may be delivered as naked RNA (unpackaged) or via deliveryvehicles. As used herein, the terms “delivery vehicle,” “transfervehicle,” “nanoparticle” or grammatical equivalent, are usedinterchangeably.

In some embodiments, mRNAs encoding an GAA protein may be delivered viaa single delivery vehicle. In some embodiments, mRNAs encoding an GAAprotein may be delivered via one or more delivery vehicles each of adifferent composition. According to various embodiments, suitabledelivery vehicles include, but are not limited to polymer basedcarriers, such as polyethyleneimine (PEI), lipid nanoparticles andliposomes, nanoliposomes, ceramide-containing nanoliposomes,proteoliposomes, both natural and synthetically-derived exosomes,natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates,calcium phosphor-silicate nanoparticulates, calcium phosphatenanoparticulates, silicon dioxide nanoparticulates, nanocrystallineparticulates, semiconductor nanoparticulates, poly(D-arginine),sol-gels, nanodendrimers, starch-based delivery systems, micelles,emulsions, niosomes, multi-domain-block polymers (vinyl polymers,polypropyl acrylic acid polymers, dynamic polyconjugates), dry powderformulations, plasmids, viruses, calcium phosphate nucleotides,aptamers, peptides and other vectorial tags.

Liposomal Delivery Vehicles

In some embodiments, a suitable delivery vehicle is a liposomal deliveryvehicle, e.g., a lipid nanoparticle. As used herein, liposomal deliveryvehicles, e.g., lipid nanoparticles, are usually characterized asmicroscopic vesicles having an interior aqua space sequestered from anouter medium by a membrane of one or more bilayers. Bilayer membranes ofliposomes are typically formed by amphiphilic molecules, such as lipidsof synthetic or natural origin that comprise spatially separatedhydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). Bilayer membranes of the liposomes can also be formed byamphophilic polymers and surfactants (e.g., polymerosomes, niosomes,etc.). In the context of the present invention, a liposomal deliveryvehicle typically serves to transport a desired mRNA to a target cell ortissue.

Cationic Lipids

In some embodiments, liposomes may comprise one or more cationic lipids.As used herein, the phrase “cationic lipid” refers to any of a number oflipid species that have a net positive charge at a selected pH, such asphysiological pH. Several cationic lipids have been described in theliterature, many of which are commercially available. Particularlysuitable cationic lipids for use in the compositions and methods of theinvention include those described in international patent publicationsWO 2010/053572 (and particularly, CI 2-200 described at paragraph[00225]) and WO 2012/170930, both of which are incorporated herein byreference. In certain embodiments, the compositions and methods of theinvention employ a lipid nanoparticles comprising an ionizable cationiclipid described in U.S. provisional patent application 61/617,468, filedMar. 29, 2012 (incorporated herein by reference), such as, e.g, (15Z,18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and(15Z,18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-5, 15, 18-trien-1-amine (HGT5002).

In some embodiments, provided liposomes include a cationic lipiddescribed in WO 2013/063468 and in U.S. provisional application entitled“Lipid Formulations for Delivery of Messenger RNA” filed concurrentlywith the present application on even date, both of which areincorporated by reference herein.

In some embodiments, a cationic lipid comprises a compound of formulaI-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each R² independently is hydrogen or C₁₋₃ alkyl;    -   each q independently is 2 to 6;    -   each R′ independently is hydrogen or C₁₋₃ alkyl;    -   and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² independently is hydrogen, methyl or ethyl.In some embodiments, each R² independently is hydrogen or methyl. Insome embodiments, each R² is hydrogen.

In some embodiments, each q independently is 3 to 6. In someembodiments, each q independently is 3 to 5. In some embodiments, each qis 4.

In some embodiments, each R′ independently is hydrogen, methyl or ethyl.In some embodiments, each R′ independently is hydrogen or methyl. Insome embodiments, each R′ independently is hydrogen.

In some embodiments, each R^(L) independently is C₈₋₁₂ alkyl. In someembodiments, each R^(L) independently is n-C₈₋₁₂ alkyl. In someembodiments, each R^(L) independently is C₉₋₁₁ alkyl. In someembodiments, each R^(L) independently is n-C₉₋₁₁ alkyl. In someembodiments, each R^(L) independently is C₁₀ alkyl. In some embodiments,each R^(L) independently is n-C₁₀ alkyl.

In some embodiments, each R² independently is hydrogen or methyl; each qindependently is 3 to 5; each R′ independently is hydrogen or methyl;and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q independently is 3 to5; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q is 4; each R′ ishydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, a cationic lipid comprises a compound of formulaI-g:

or a pharmaceutically acceptable salt thereof, wherein each R^(L)independently is C₈₋₁₂ alkyl. In some embodiments, each R^(L)independently is n-C₈₋₁₂ alkyl. In some embodiments, each R^(L)independently is C₉₋₁₁ alkyl. In some embodiments, each R^(L)independently is n-C₉₋₁₁ alkyl. In some embodiments, each R^(L)independently is C₁₀ alkyl. In some embodiments, each R^(L) is n-C₁₀alkyl.

In particular embodiments, provided liposomes include a cationic lipidcKK-E12, or(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).Structure of cKK-E12 is shown below:

In some embodiments, the one or more cationic lipids may beN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA”(Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No.4,897,355). DOTMA can be formulated alone or can be combined with theneutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or othercationic or non-cationic lipids into a liposomal transfer vehicle or alipid nanoparticle, and such liposomes can be used to enhance thedelivery of nucleic acids into target cells. Other suitable cationiclipids include, for example, 5-carboxyspermylglycinedioctadecylamide or“DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.

Additional exemplary cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′,1-2′-octadecadienoxy)propane or “CpLinDMA”,N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin--DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In someembodiments, one or more of the cationic lipids comprise at least one ofan imidazole, dialkylamino, or guanidinium moiety.

In some embodiments, the one or more cationic lipids may be chosen fromXTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889,the teachings of which are incorporated herein by reference in theirentirety), ICE (WO 2011/068810, the teachings of which are incorporatedherein by reference in their entirety), HGT5000 (U.S. Provisional PatentApplication No. 61/617,468, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001 (cis or trans)(Provisional Patent Application No. 61/617,468), aminoalcohol lipidoidssuch as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S.C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

In some embodiments, the percentage of cationic lipid in a liposome maybe greater than 10%, greater than 20%, greater than 30%, greater than40%, greater than 50%, greater than 60%, or greater than 70%. In someembodiments, cationic lipid(s) constitute(s) about 30-50% (e.g., about30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) ofthe liposome by weight. In some embodiments, the cationic lipid (e.g.,cKK-E12) constitutes about 30%, about 35%, about 40%, about 45%, orabout 50% of the liposome by molar ratio.

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic(“helper”) lipids. As used herein, the phrase “non-cationic lipid”refers to any neutral, zwitterionic or anionic lipid. As used herein,the phrase “anionic lipid” refers to any of a number of lipid speciesthat carry a net negative charge at a selected H, such as physiologicalpH. Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. In some embodiments, the non-cationic lipid maycomprise a molar ratio of about 5% to about 90%, or about 10% to about70% of the total lipid present in a liposome. In some embodiments, anon-cationic lipid is a neutral lipid, i.e., a lipid that does not carrya net charge in the conditions under which the composition is formulatedand/or administered. In some embodiments, the percentage of non-cationiclipid in a liposome may be greater than 5%, greater than 10%, greaterthan 20%, greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or morecholesterol-based lipids. For example, suitable cholesterol-basedcationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE. In some embodiments, thecholesterol-based lipid may comprise a molar ration of about 2% to about30%, or about 5% to about 20% of the total lipid present in a liposome.In some embodiments, The percentage of cholesterol-based lipid in thelipid nanoparticle may be greater than 5, %, 10%, greater than 20%,greater than 30%, or greater than 40%.

PEGylated Lipids

In some embodiments, provided liposomes comprise one or more PEGylatedlipids. For example, the use of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized ceramides(PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplatedby the present invention in combination with one or more of the cationicand, in some embodiments, other lipids together which comprise theliposome. Contemplated PEG-modified lipids include, but are not limitedto, a polyethylene glycol chain of up to 5 kDa in length covalentlyattached to a lipid with alkyl chain(s) of C₆-C₂₀ length. In someembodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterolor PEG-2K. The addition of such components may prevent complexaggregation and may also provide a means for increasing circulationlifetime and increasing the delivery of the lipid-nucleic acidcomposition to the target cell, (Klibanov et al. (1990) FEBS Letters,268 (1): 235-237), or they may be selected to rapidly exchange out ofthe formulation in vivo (see U.S. Pat. No. 5,885,613).

In some embodiments, particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C₁₄ or C₁₈). ThePEG-modified phospholipid and derivitized lipids of the presentinvention may comprise a molar ratio from about 0% to about 15%, about0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, orabout 2% of the total lipid present in the liposome.

According to various embodiments, the selection of cationic lipids,non-cationic lipids and/or PEG-modified lipids which comprise the lipidnanoparticle, as well as the relative molar ratio of such lipids to eachother, is based upon the characteristics of the selected lipid(s), thenature of the intended target cells, the characteristics of the mRNA tobe delivered. Additional considerations include, for example, thesaturation of the alkyl chain, as well as the size, charge, pH, pKa,fusogenicity and toxicity of the selected lipid(s). Thus the molarratios may be adjusted accordingly.

Polymers

In some embodiments, a suitable delivery vehicle is formulated using apolymer as a carrier, alone or in combination with other carriersincluding various lipids described herein. Thus, in some embodiments,liposomal delivery vehicles, as used herein, also encompass polymercontaining nanoparticles. Suitable polymers may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine(PEI). When PEI is present, it may be branched PEI of a molecular weightranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

A suitable liposome for the present invention may include one or more ofany of the cationic lipids, non-cationic lipids, cholesterol lipids,PEGylated lipids and/or polymers described herein at various ratios. Asnon-limiting examples, a suitable liposome formulation may include acombination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K;C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol andDMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K.

In various embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE,and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%) of the liposome by molar ratio. In some embodiments, thepercentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/orHGT4003) is or greater than about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, or about 60% of the liposome by molar ratio.

In some embodiments, the ratio of cationic lipid(s) to non-cationiclipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) may bebetween about 30-60:25-35:20-30:1-15, respectively. In some embodiments,the ratio of cationic lipid(s) to non-cationic lipid(s) tocholesterol-based lipid(s) to PEGylated lipid(s) is approximately40:30:20:10, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEGylated lipid(s) is approximately 40:30:25:5, respectively. In someembodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) tocholesterol-based lipid(s) to PEGylated lipid(s) is approximately40:32:25:3, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEGylated lipid(s) is approximately 50:25:20:5.

Synthesis of mRNA

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence fordesired mRNA and a termination signal.

Desired mRNA sequence(s) according to the invention may be determinedand incorporated into a DNA template using standard methods. Forexample, starting from a desired amino acid sequence (e.g., an enzymesequence), a virtual reverse translation is carried out based on thedegenerated genetic code. Optimization algorithms may then be used forselection of suitable codons. Typically, the G/C content can beoptimized to achieve the highest possible G/C content on one hand,taking into the best possible account the frequency of the tRNAsaccording to codon usage on the other hand. The optimized RNA sequencecan be established and displayed, for example, with the aid of anappropriate display device and compared with the original (wild-type)sequence. A secondary structure can also be analyzed to calculatestabilizing and destabilizing properties or, respectively, regions ofthe RNA.

Modified mRNA

In some embodiments, mRNA according to the present invention may besynthesized as unmodified or modified mRNA. Typically, mRNAs aremodified to enhance stability. Modifications of mRNA can include, forexample, modifications of the nucleotides of the RNA. An modified mRNAaccording to the invention can thus include, for example, backbonemodifications, sugar modifications or base modifications. In someembodiments, mRNAs may be synthesized from naturally occurringnucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purines (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g., from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732,4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418,5,153,319, 5,262,530 and 5,700,642, the disclosures of which areincorporated by reference in their entirety.

In some embodiments, mRNAs (e.g., GAA-encoding mRNAs) may contain RNAbackbone modifications. Typically, a backbone modification is amodification in which the phosphates of the backbone of the nucleotidescontained in the RNA are modified chemically. Exemplary backbonemodifications typically include, but are not limited to, modificationsfrom the group consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g. cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., GAA-encoding mRNAs) may contain sugarmodifications. A typical sugar modification is a chemical modificationof the sugar of the nucleotides it contains including, but not limitedto, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-T-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(T-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates(2′-azido-T-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate).

In some embodiments, mRNAs (e.g., GAA-encoding mRNAs) may containmodifications of the bases of the nucleotides (base modifications). Amodified nucleotide which contains a base modification is also called abase-modified nucleotide. Examples of such base-modified nucleotidesinclude, but are not limited to, 2-amino-6-chloropurine riboside5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5 ‘-triphosphate, 7-deazaguanosine 5’-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine5′-triphosphate, 06-methylguanosine 5′-triphosphate, pseudouridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., GAA-encoding mRNAs) include a 5′cap structure. A 5′ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups fromthe 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, mRNAs (e.g., GAA-encoding mRNAs) include a 3′poly(A) tail structure. A poly-A tail on the 3′ terminus of mRNAtypically includes about 10 to 300 adenosine nucleotides (SEQ ID NO: 4)(e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosinenucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In someembodiments, mRNAs include a 3′ poly(C) tail structure. A suitablepoly-C tail on the 3′ terminus of mRNA typically include about 10 to 200cytosine nucleotides (SEQ ID NO: 5) (e.g., about 10 to 150 cytosinenucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10to 40 cytosine nucleotides). The poly-C tail may be added to the poly-Atail or may substitute the poly-A tail.

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Cap Structure

In some embodiments, mRNAs include a 5′ cap structure. A 5′ cap istypically added as follows: first, an RNA terminal phosphatase removesone of the terminal phosphate groups from the 5′ nucleotide, leaving twoterminal phosphates; guanosine triphosphate (GTP) is then added to theterminal phosphates via a guanylyl transferase, producing a 5′5′5triphosphate linkage; and the 7-nitrogen of guanine is then methylatedby a methyltransferase. Examples of cap structures include, but are notlimited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

Naturally occurring cap structures comprise a 7-methyl guanosine that islinked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in a dinucleotide cap of m⁷G(5′)ppp(5′)N, where Nis any nucleoside. In vivo, the cap is added enzymatically. The cap isadded in the nucleus and is catalyzed by the enzyme guanylyltransferase. The addition of the cap to the 5′ terminal end of RNAoccurs immediately after initiation of transcription. The terminalnucleoside is typically a guanosine, and is in the reverse orientationto all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription ism⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap intranscription with T7 or SP6 RNA polymerase in vitro to obtain RNAshaving a cap structure in their 5′-termini. The prevailing method forthe in vitro synthesis of capped mRNA employs a pre-formed dinucleotideof the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator oftranscription.

To date, a usual form of a synthetic dinucleotide cap used in in vitrotranslation experiments is the Anti-Reverse Cap Analog (“ARCA”) ormodified ARCA, which is generally a modified cap analog in which the 2′or 3′ OH group is replaced with —OCH₃.

Additional cap analogs include, but are not limited to, a chemicalstructures selected from the group consisting of m⁷GpppG, m⁷GpppA,m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog(e.g., m²′⁷GpppG), trimethylated cap analog (e.g., m^(2,2,7)Gppp),dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reversecap analogs (e.g., ARCA; m^(7, 2′Ome)GpppG, m^(72′d)GpppG,m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives)(see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs withsuperior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”)linked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. Apreferred embodiment of a m⁷G cap utilized in embodiments of theinvention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a2′-O-methyl residue of the ribose attached to bases 1 and 2. In someembodiments, the cap is a Cap1 structure. Cap1 structures have a2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2structure. Cap2 structures have a 2′-O-methyl residue attached to bothbases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which arecommercially available. These include the m⁷GpppG described above, aswell as the ARCA 3′-OCH₃ and 2′—OCH₃ cap analogs (Jemielity, J. et al.,RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodimentsof the invention include N7-benzylated dinucleoside tetraphosphateanalogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),phosphorothioate cap analogs (described in Grudzien-Nogalska, E., etal., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylatedcap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529,incorporated by reference herein.

Tail Structure

Typically, the presence of a “tail” serves to protect the mRNA fromexonuclease degradation. The poly A tail is thought to stabilize naturalmessengers and synthetic sense RNA. Therefore, in certain embodiments along poly A tail can be added to an mRNA molecule thus rendering the RNAmore stable. Poly A tails can be added using a variety of art-recognizedtechniques. For example, long poly A tails can be added to synthetic orin vitro transcribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, mRNAs include a 3′ poly(A) tail structure.Typically, the length of the poly A tail can be at least about 10, 50,100, 200, 300, 400 at least 500 nucleotides (SEQ ID NO: 6). In someembodiments, a poly-A tail on the 3′ terminus of mRNA typically includesabout 10 to 300 adenosine nucleotides (SEQ ID NO: 4) (e.g., about 10 to200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides,or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAsinclude a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′terminus of mRNA typically include about 10 to 200 cytosine nucleotides(SEQ ID NO: 5) (e.g., about 10 to 150 cytosine nucleotides, about 10 to100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). Thepoly-C tail may be added to the poly-A tail or may substitute the poly-Atail.

In some embodiments, the length of the poly A or poly C tail is adjustedto control the stability of a modified sense mRNA molecule of theinvention and, thus, the transcription of protein. For example, sincethe length of the poly A tail can influence the half-life of a sensemRNA molecule, the length of the poly A tail can be adjusted to modifythe level of resistance of the mRNA to nucleases and thereby control thetime course of polynucleotide expression and/or polypeptide productionin a target cell.

5′ and 3′ Untranslated Region

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA moleculeswhich are stable (e.g., globin, actin, GAPDH, tubulin, histone, orcitric acid cycle enzymes) to increase the stability of the sense mRNAmolecule. For example, a 5′ UTR sequence may include a partial sequenceof a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improvethe nuclease resistance and/or improve the half-life of thepolynucleotide. Also contemplated is the inclusion of a sequenceencoding human growth hormone (hGH), or a fragment thereof to the 3′ endor untranslated region of the polynucleotide (e.g., mRNA) to furtherstabilize the polynucleotide. Generally, these modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of thepolynucleotide relative to their unmodified counterparts, and include,for example modifications made to improve such polynucleotides'resistance to in vivo nuclease digestion.

Formation of Liposomes

The liposomal transfer vehicles for use in the compositions of theinvention can be prepared by various techniques which are presentlyknown in the art. The liposomes for use in provided compositions can beprepared by various techniques which are presently known in the art. Forexample, multilamellar vesicles (MLV) may be prepared according toconventional techniques, such as by depositing a selected lipid on theinside wall of a suitable container or vessel by dissolving the lipid inan appropriate solvent, and then evaporating the solvent to leave a thinfilm on the inside of the vessel or by spray drying. An aqueous phasemay then added to the vessel with a vortexing motion which results inthe formation of MLVs. Unilamellar vesicles (ULV) can then be formed byhomogenization, sonication or extrusion of the multilamellar vesicles.In addition, unilamellar vesicles can be formed by detergent removaltechniques.

In certain embodiments, provided compositions comprise a liposomewherein the mRNA is associated on both the surface of the liposome andencapsulated within the same liposome. For example, during preparationof the compositions of the present invention, cationic liposomes mayassociate with the mRNA through electrostatic interactions. For example,during preparation of the compositions of the present invention,cationic liposomes may associate with the mRNA through electrostaticinteractions.

In some embodiments, the compositions and methods of the inventioncomprise mRNA encapsulated in a liposome. In some embodiments, the oneor more mRNA species may be encapsulated in the same liposome. In someembodiments, the one or more mRNA species may be encapsulated indifferent liposomes. In some embodiments, the mRNA is encapsulated inone or more liposomes, which differ in their lipid composition, molarratio of lipid components, size, charge (Zeta potential), targetingligands and/or combinations thereof. In some embodiments, the one ormore liposome may have a different composition of cationic lipids,neutral lipid, PEG-modified lipid and/or combinations thereof. In someembodiments the one or more liposomes may have a different molar ratioof cationic lipid, neutral lipid, cholesterol and PEG-modified lipidused to create the liposome.

The process of incorporation of a desired mRNA into a liposome is oftenreferred to as “loading”. Exemplary methods are described in Lasic, etal., FEBS Lett., 312: 255-258, 1992, which is incorporated herein byreference. The liposome-incorporated nucleic acids may be completely orpartially located in the interior space of the liposome, within thebilayer membrane of the liposome, or associated with the exteriorsurface of the liposome membrane. The incorporation of a nucleic acidinto liposomes is also referred to herein as “encapsulation” wherein thenucleic acid is entirely contained within the interior space of theliposome. The purpose of incorporating a mRNA into a transfer vehicle,such as a liposome, is often to protect the nucleic acid from anenvironment which may contain enzymes or chemicals that degrade nucleicacids and/or systems or receptors that cause the rapid excretion of thenucleic acids. Accordingly, in some embodiments, a suitable deliveryvehicle is capable of enhancing the stability of the mRNA containedtherein and/or facilitate the delivery of mRNA to the target cell ortissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be madein various sizes. In some embodiments, provided liposomes may be madesmaller than previously known mRNA encapsulating liposomes. In someembodiments, decreased size of liposomes is associated with moreefficient delivery of mRNA. Selection of an appropriate liposome sizemay take into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made.

In some embodiments, an appropriate size of liposome is selected tofacilitate systemic distribution of antibody encoded by the mRNA. Insome embodiments, it may be desirable to limit transfection of the mRNAto certain cells or tissues. For example, to target hepatocytes aliposome may be sized such that its dimensions are smaller than thefenestrations of the endothelial layer lining hepatic sinusoids in theliver; in such cases the liposome could readily penetrate suchendothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that thedimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,a liposome may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the liposome particle. In some embodiments, asuitable liposome has a size no greater than about 250 nm (e.g., nogreater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75nm, or 50 nm). In some embodiments, a suitable liposome has a sizeranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm).In some embodiments, a suitable liposome has a size ranging from about100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm,100-150 nm). In some embodiments, a suitable liposome has a size rangingfrom about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70nm, 10-60 nm, or 10-50 nm). In a particular embodiment, a suitableliposome has a size less than about 100 nm.

A variety of alternative methods known in the art are available forsizing of a population of liposomes. One such sizing method is describedin U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicatinga liposome suspension either by bath or probe sonication produces aprogressive size reduction down to small ULV less than about 0.05microns in diameter. Homogenization is another method that relies onshearing energy to fragment large liposomes into smaller ones. In atypical homogenization procedure, MLV are recirculated through astandard emulsion homogenizer until selected liposome sizes, typicallybetween about 0.1 and 0.5 microns, are observed. The size of theliposomes may be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981),incorporated herein by reference. Average liposome diameter may bereduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Pharmaceutical Compositions

To facilitate expression of mRNA in vivo, delivery vehicles such asliposomes can be formulated in combination with one or more additionalnucleic acids, carriers, targeting ligands or stabilizing reagents, orin pharmacological compositions where it is mixed with suitableexcipients. Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

Provided liposomally-encapsulated or associated mRNAs, and compositionscontaining the same, may be administered and dosed in accordance withcurrent medical practice, taking into account the clinical condition ofthe subject, the site and method of administration, the scheduling ofadministration, the subject's age, sex, body weight and other factorsrelevant to clinicians of ordinary skill in the art. The “effectiveamount” for the purposes herein may be determined by such relevantconsiderations as are known to those of ordinary skill in experimentalclinical research, pharmacological, clinical and medical arts. In someembodiments, the amount administered is effective to achieve at leastsome stabilization, improvement or elimination of symptoms and otherindicators as are selected as appropriate measures of disease progress,regression or improvement by those of skill in the art. For example, asuitable amount and dosing regimen is one that causes at least transientprotein (e.g., enzyme) production.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, or intranasal. In particular embodiments,the intramuscular administration is to a muscle selected from the groupconsisting of skeletal muscle, smooth muscle and cardiac muscle. In someembodiments the administration results in delivery of the mRNA to amuscle cell. In some embodiments the administration results in deliveryof the mRNA to a hepatocyte (i.e., liver cell). In a particularembodiment, the intramuscular administration results in delivery of themRNA to a muscle cell.

Alternatively or additionally, liposomally encapsulated mRNAs andcompositions of the invention may be administered in a local rather thansystemic manner, for example, via injection of the pharmaceuticalcomposition directly into a targeted tissue, preferably in a sustainedrelease formulation. Local delivery can be affected in various ways,depending on the tissue to be targeted. For example, aerosols containingcompositions of the present invention can be inhaled (for nasal,tracheal, or bronchial delivery); compositions of the present inventioncan be injected into the site of injury, disease manifestation, or pain,for example; compositions can be provided in lozenges for oral,tracheal, or esophageal application; can be supplied in liquid, tabletor capsule form for administration to the stomach or intestines, can besupplied in suppository form for rectal or vaginal application; or caneven be delivered to the eye by use of creams, drops, or even injection.Formulations containing provided compositions complexed with therapeuticmolecules or ligands can even be surgically administered, for example inassociation with a polymer or other structure or substance that canallow the compositions to diffuse from the site of implantation tosurrounding cells. Alternatively, they can be applied surgically withoutthe use of polymers or supports.

Provided methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of thetherapeutic agents (e.g., mRNA encoding a GAA protein) described herein.Therapeutic agents can be administered at regular intervals, dependingon the nature, severity and extent of the subject's condition (e.g.,Pompe disease). In some embodiments, a therapeutically effective amountof the therapeutic agents (e.g., mRNA encoding a GAA protein) of thepresent invention may be administered intrathecally periodically atregular intervals (e.g., once every year, once every six months, onceevery five months, once every three months, bimonthly (once every twomonths), monthly (once every month), biweekly (once every two weeks),twice a month, once every 30 days, once every 28 days, once every 14days, once every 10 days, once every 7 days, weekly, twice a week, dailyor continuously).

In some embodiments, provided liposomes and/or compositions areformulated such that they are suitable for extended-release of the mRNAcontained therein. Such extended-release compositions may beconveniently administered to a subject at extended dosing intervals. Forexample, in one embodiment, the compositions of the present inventionare administered to a subject twice a day, daily or every other day. Ina preferred embodiment, the compositions of the present invention areadministered to a subject twice a week, once a week, once every 7 days,once every 10 days, once every 14 days, once every 28 days, once every30 days, once every two weeks, once every three weeks, or morepreferably once every four weeks, once a month, twice a month, onceevery six weeks, once every eight weeks, once every other month, onceevery three months, once every four months, once every six months, onceevery eight months, once every nine months or annually. Alsocontemplated are compositions and liposomes which are formulated fordepot administration (e.g., intramuscularly, subcutaneously,intravitreally) to either deliver or release a mRNA over extendedperiods of time. Preferably, the extended-release means employed arecombined with modifications made to the mRNA to enhance stability.

As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating Pompe disease). For example, a therapeuticallyeffective amount may be an amount sufficient to achieve a desiredtherapeutic and/or prophylactic effect. Generally, the amount of atherapeutic agent (e.g., mRNA encoding a GAA protein) administered to asubject in need thereof will depend upon the characteristics of thesubject. Such characteristics include the condition, disease severity,general health, age, sex and body weight of the subject. One of ordinaryskill in the art will be readily able to determine appropriate dosagesdepending on these and other related factors. In addition, bothobjective and subjective assays may optionally be employed to identifyoptimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific protein employed; the duration of the treatment; and likefactors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg body weight to 500 mg/kg body weight, e.g., from about0.005 mg/kg body weight to 400 mg/kg body weight, from about 0.005 mg/kgbody weight to 300 mg/kg body weight, from about 0.005 mg/kg body weightto 200 mg/kg body weight, from about 0.005 mg/kg body weight to 100mg/kg body weight, from about 0.005 mg/kg body weight to 90 mg/kg bodyweight, from about 0.005 mg/kg body weight to 80 mg/kg body weight, fromabout 0.005 mg/kg body weight to 70 mg/kg body weight, from about 0.005mg/kg body weight to 60 mg/kg body weight, from about 0.005 mg/kg bodyweight to 50 mg/kg body weight, from about 0.005 mg/kg body weight to 40mg/kg body weight, from about 0.005 mg/kg body weight to 30 mg/kg bodyweight, from about 0.005 mg/kg body weight to 25 mg/kg body weight, fromabout 0.005 mg/kg body weight to 20 mg/kg body weight, from about 0.005mg/kg body weight to 15 mg/kg body weight, from about 0.005 mg/kg bodyweight to 10 mg/kg body weight.

In some embodiments, the therapeutically effective dose is greater thanabout 0.1 mg/kg body weight, greater than about 0.5 mg/kg body weight,greater than about 1.0 mg/kg body weight, greater than about 3 mg/kgbody weight, greater than about 5 mg/kg body weight, greater than about10 mg/kg body weight, greater than about 15 mg/kg body weight, greaterthan about 20 mg/kg body weight, greater than about 30 mg/kg bodyweight, greater than about 40 mg/kg body weight, greater than about 50mg/kg body weight, greater than about 60 mg/kg body weight, greater thanabout 70 mg/kg body weight, greater than about 80 mg/kg body weight,greater than about 90 mg/kg body weight, greater than about 100 mg/kgbody weight, greater than about 150 mg/kg body weight, greater thanabout 200 mg/kg body weight, greater than about 250 mg/kg body weight,greater than about 300 mg/kg body weight, greater than about 350 mg/kgbody weight, greater than about 400 mg/kg body weight, greater thanabout 450 mg/kg body weight, greater than about 500 mg/kg body weight.In a particular embodiment, the therapeutically effective dose is 1.0mg/kg. In some embodiments, the therapeutically effective dose of 1.0mg/kg is administered intramuscularly or intravenously.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomes disclosed herein and relatedmethods for the use of such compositions as disclosed for example, inU.S. Provisional Application No. 61/494,882, filed Jun. 8, 2011, theteachings of which are incorporated herein by reference in theirentirety. For example, lyophilized pharmaceutical compositions accordingto the invention may be reconstituted prior to administration or can bereconstituted in vivo. For example, a lyophilized pharmaceuticalcomposition can be formulated in an appropriate dosage form (e.g., anintradermal dosage form such as a disk, rod or membrane) andadministered such that the dosage form is rehydrated over time in vivoby the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desiredtissue. In some embodiments, the GAA mRNA delivered by providedliposomes or compositions is expressed in the tissue in which theliposomes and/or compositions were administered. In some embodiments,the mRNA delivered is expressed in a tissue different from the tissue inwhich the liposomes and/or compositions were administered. Exemplarytissues in which delivered mRNA may be delivered and/or expressedinclude, but are not limited to the liver, kidney, heart, spleen, serum,brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.

In some embodiments, administering the provided composition results inan increased GAA mRNA expression level in a biological sample from asubject as compared to a baseline expression level before treatment.Typically, the baseline level is measured immediately before treatment.Biological samples include, for example, whole blood, serum, plasma,urine and tissue samples (e.g., muscle, liver, skin fibroblasts). Insome embodiments, administering the provided composition results in anincreased GAA mRNA expression level by at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline levelimmediately before treatment. In some embodiments, administering theprovided composition results in an increased GAA mRNA expression levelas compared to a GAA mRNA expression level in subjects who are nottreated

According to the present invention, a therapeutically effective dose ofthe provided composition, when administered regularly, results in anincreased hepatic GAA protein level in a subject as compared to abaseline hepatic GAA protein level before treatment. In someembodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in an increased muscleGAA protein level in a subject as compared to a baseline muscle GAAprotein level before treatment. In some embodiments, the muscle isskeletal muscle (e.g., striated muscle, voluntary muscle), smooth muscle(e.g., visceral muscle, involuntary muscle) or cardiac muscle. In someembodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in a reduced serumcreatine kinase level in a subject as compared to a baseline creatinekinase level before treatment. In some embodiments, a therapeuticallyeffective dose of the provided composition, when administered regularly,results in a reduced urinary glucose tetrasaccharide,(Glcα1-6Glcα1-4Glcα1-4Glc or Glc₄) level in a subject as compared to abaseline Glc₄ level before treatment. In some embodiments, atherapeutically effective dose of the provided composition, whenadministered regularly, results in a reduced serum aspartatetransaminase (e.g., AST, aspartate aminotransferase, serum, glutamicoxaloacetic transaminase) level in as subject as compared to a baselineAST level before treatment. In some embodiments, a therapeuticallyeffective dose of the provided composition, when administered regularly,results in a reduced serum alanine transaminase (e.g., ALT, alanineaminotransferase, serum glutamic-pyruvic transaminase) level in asubject as compared to a baseline ALT level before treatment. In someembodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in a reduced serumlactate dehydrogenase (e.g., LDH, lactic dehydrogenase) level in asubject as compared to a baseline LDH level before treatment. In someembodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in an increased GAAenzyme activity level in a biological sample from a subject as comparedto a baseline GAA enzyme activity level before treatment.

In some embodiments, administering the provided composition results inan increased GAA protein level in the liver of a subject as compared toa baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in the liver by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in the liver as compared to aGAA protein level in the liver of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased GAA protein level in skeletal muscle of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in skeletal muscle by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in skeletal muscle as comparedto the GAA protein level in skeletal muscle of subjects who are nottreated.

In some embodiments, administering the provided composition results inan increased GAA protein level in cardiac muscle of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in cardiac muscle by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in cardiac muscle as comparedto a GAA protein level in cardiac muscle of subjects who are nottreated.

In some embodiments, administering the provided composition results inan increased level of GAA protein in smooth muscle of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in smooth muscle by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in smooth muscle as comparedto a GAA protein level in smooth muscle of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased level of GAA protein in a muscle cell of a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments themuscle cell is a myocyte, a myotube, a myoblast, a cardiomyocyte or acardiomyoblast. In some embodiments, administering the providedcomposition results in an increased GAA protein level in the muscle cellby at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in a muscle cell as compared to the GAA protein level amuscle cell of subjects who are not treated.

In some embodiments, administering the provided composition results inan increased level of GAA protein in a liver cell (e.g., a hepatocyte)of a subject as compared to a baseline level before treatment.Typically, the baseline level is measured immediately before treatment.In some embodiments, administering the provided composition results inan increased GAA protein level in the liver cell by at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baselinelevel before treatment. In some embodiments, administering the providedcomposition results in an increased GAA protein level in a liver cell ascompared to the GAA protein level a liver cell of subjects who are nottreated.

In some embodiments, administering the provided composition results inan increased GAA protein level in plasma or serum of subject as comparedto a baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in an increased GAAprotein level in plasma or serum by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA protein level in plasma or serum as comparedto a GAA protein level in plasma or serum of subjects who are nottreated.

In some embodiments, administering the provided composition results inreduced a serum creatine kinase level in a subject as compared to abaseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serumcreatine kinase level by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% as compared to a baseline serum creatine kinaselevel immediately before treatment. In some embodiments, administeringof the provided composition results in a reduced serum creatine kinaselevel to less than about 2000 IU/L, 1500 IU/L, 1000 IU/L, 750 IU/L, 500IU/L, 250 IU/L, 100 IU/L, 90 IU/L, 80 IU/L, 70 IU/L or 60 IU/L. In someembodiments, administering the provided composition results in a reducedserum creatine kinase level as compared to a serum creatine kinase levelin subjects who are not treated.

In some embodiments, administering the provided composition results in areduced urinary Glc₄ level in a subject as compared to a baseline levelbefore treatment. Typically, the baseline level is measured immediatelybefore treatment. In some embodiments, administering the providedcomposition results in a reduced urinary Glc₄ level by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to abaseline level immediately before treatment. In some embodiments,administering the provided composition results in a reduced urinary Glc₄level to less than about 100 mmol Glc₄/mol creatinine, 90 mmol Glc₄/molcreatinine, 80 mmol Glc₄/mol creatinine, 70 mmol Glc₄/mol creatinine, 60mmol Glc₄/mol creatinine, 50 mmol Glc₄/mol creatinine, 40 mmol Glc₄/molcreatinine, 30 mmol Glc₄/mol creatinine or 20 mmol Glc₄/mol creatinine.In some embodiments, administering the provided composition results in areduced urinary Glc₄ level as compared to a urinary Glc₄ level insubjects who are not treated.

In some embodiments, administering the provided composition results in areduced muscle glycogen level in a subject as compared to a baselinelevel before treatment. Typically, a baseline level is measuredimmediately before treatment. In some embodiments, administering theprovided composition results in a reduced muscle glycogen level by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level immediately before treatment. In someembodiments, administering the provided composition results in a reducedmuscle glycogen level as compared to a muscle glycogen level in subjectswho are not treated. In particular embodiments, the muscle is skeletalmuscle, smooth muscle or cardiac muscle.

In some embodiments, administering the provided composition results in areduced liver glycogen level in a subject as compared to a baselinelevel before treatment. Typically, the baseline level is measuredimmediately before treatment. In some embodiments, administering theprovided composition results in a reduced liver glycogen level by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to a baseline level immediately before treatment. In someembodiments, administering the provided composition results in a reducedliver glycogen level as compared to a liver glycogen level in subjectswho are not treated.

In some embodiments, administering the provided composition results in areduced serum aspartate transaminase (AST) level in a subject ascompared to a baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serum ASTlevel by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or95% as compared to a baseline level immediately before treatment. Insome embodiments, administering the provided composition results in areduced serum AST level to less than about 600 IU/L, 500 IU/L, 400 IU/L,300 IU/L, 200 IU/L, 100 IU/L, 50 IU/L, 25 IU/L, 20 IU/L or 10 IU/L. Insome embodiments, administering the provided composition results in areduced serum AST level as compared to a serum AST level in subjects whoare not treated.

In some embodiments, administering the provided composition results in areduced serum alanine transaminase (ALT) level in a subject as comparedto a baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serum ALTlevel by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or95% as compared to a baseline level immediately before treatment. Insome embodiments, administering the provided composition results in areduced serum ALT level to less than about 1000 IU/L, 900 IU/L, 800IU/L, 700 IU/L, 600 IU/L, 500 IU/L, 400 IU/L, 300 IU/L, 200 IU/L, 100IU/L, 50 IU/L, 25 IU/L, 20 IU/L or 10 IU/L. In some embodiments,administering the provided composition results in a reduced serum ALTlevel as compared to a serum ALT level in subjects who are not treated.

In some embodiments, administering the provided composition results in areduced serum lactate dehydrogenase (LDH) level in a subject as comparedto a baseline level before treatment. Typically, the baseline level ismeasured immediately before treatment. In some embodiments,administering the provided composition results in a reduced serumlactate dehydrogenase LDH level by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline levelimmediately before treatment. In some embodiments, administering theprovided composition results in a reduced serum LDH level to less thanabout 2000 IU/L, 1500 IU/L, 1000 IU/L, 900 IU/L, 800 IU/L, 700 IU/L, 600IU/L, 500 IU/L, 400 IU/L, 300 IU/L, 200 IU/L or 100 IU/L. In someembodiments, administering the provided composition results in a reducedserum LDH level as compared to a serum LDH level in subjects who are nottreated.

In some embodiments, administering the provided composition results inincreased GAA enzyme activity in a biological sample from a subject ascompared to the baseline level before treatment. Typically, the baselinelevel is measured immediately before treatment. Biological samplesinclude, for example, whole blood, serum, plasma, urine and tissuesamples (e.g., muscle, liver, skin fibroblasts). In some embodiments,administering the provided composition results in an increased GAAenzyme activity by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95% as compared to a baseline level immediately beforetreatment. In some embodiments, administering the provided compositionresults in an increased GAA enzyme activity as compared to GAA enzymeactivity in subjects who are not treated.

According to various embodiments, the timing of expression of deliveredmRNAs can be tuned to suit a particular medical need. In someembodiments, the expression of the protein encoded by delivered mRNA isdetectable 1, 2, 3, 6, 12, 24, 48, 72, and/or 96 hours afteradministration of provided liposomes and/or compositions. In someembodiments, the expression of the protein encoded by delivered mRNA isdetectable 1 week, two weeks, and/or 1 month after administration.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same.

Example 1. Exemplary Liposome Formulations for GAA mRNA Delivery andExpression

This example provides exemplary liposome formulations for effectivedelivery and expression of GAA mRNA in vivo.

Lipid Materials

The formulations described herein include a multi-component lipidmixture of varying ratios employing one or more cationic lipids, helperlipids (e.g., non-cationic lipids and/or cholesterol-based lipids) andPEGylated lipids designed to encapsulate mRNA encoding GAA protein.Cationic lipids can include (but not exclusively) DOTAP (1,2-dioleyltrimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane),DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationiclipid saturation influences intracellular delivery of encapsulatednucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple,S.C. et al. “Rational Design of Cationic Lipids for siRNA Delivery”Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al.“Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010,107, 1864-1869), cKK-E12(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione),HGT5000, HGT5001, HGT4003, ICE, dialkylamino-based, imidazole-based,guanidinium-based, etc. Helper lipids can include (but not exclusively)DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.The PEGylated lipids can include (but not exclusively) a poly(ethylene)glycol chain of up to 5 kDa in length covalently attached to a lipidwith alkyl chain(s) of C₆-C₂₀ length.

Codon-optimized human acid alpha-glucosidase (GAA) messenger RNA wassynthesized by in vitro transcription from a plasmid DNA templateencoding the gene, which was followed by the addition of a 5′ capstructure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA capstructures by viral and cellular proteins” J. Gen. Virology 2005, 86,1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides inlength (SEQ ID NO: 7) as determined by gel electrophoresis. 5′ and 3′untranslated regions present in each mRNA product are represented as Xand Y, respectively and defined as stated (vide infra).

Exemplary Codon-Optimized Human Acid Alpha-Glucosidase (GAA) mRNAs

Construct design: X-SEQ ID NO: 3-Y. 5′ and 3′ UTR SequencesX (5′ UTR Sequence)= [SEQ ID NO: 8]GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG Y (3′ UTR Sequence)=[SEQ ID NO: 9] CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCA UCAAGCU OR[SEQ ID NO: 10] GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAU CAAAGCU

An exemplary codon-optimized human GAA mRNA sequence includes SEQ ID NO:3 described in the detailed description section.

An exemplary full-length codon-optimized human acid alpha-glucosidase(GAA) messenger RNA sequence is shown below:

[SEQ ID NO: 11] GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGAGUCAGACACCCGCCGUGCUCGCACAGGCUUCUGGCCGUGUGCGCACUCGUGAGUCUGGCGACUGCUGCGUUGCUGGGGCACAUUCUUCUCCACGACUUUCUCUUGGUGCCCCGAGAAUUGUCGGGCUCGUCGCCGGUACUGGAAGAAACCCACCCCGCACAUCAGCAGGGCGCGUCGCGGCCUGGUCCGAGGGAUGCCCAGGCACAUCCCGGAAGGCCACGAGCCGUCCCGACUCAAUGUGACGUACCUCCCAAUUCCCGGUUCGACUGUGCGCCAGACAAGGCAAUCACGCAAGAGCAGUGCGAAGCCCGUGGAUGCUGCUAUAUUCCGGCGAAGCAGGGACUUCAGGGAGCCCAGAUGGGGCAGCCCUGGUGUUUCUUCCCGCCUUCCUAUCCCUCAUAUAAGCUGGAGAAUUUGUCGUCCUCGGAAAUGGGGUAUACCGCUACUCUUACGAGAACCACCCCCACAUUCUUUCCGAAGGACAUCCUUACUCUGCGGCUCGACGUGAUGAUGGAGACAGAAAAUAGGCUGCAUUUCACGAUCAAAGACCCGGCGAACCGGAGAUAUGAGGUUCCGCUUGAGACUCCCCACGUUCACUCUCGUGCGCCUUCACCCUUGUACUCCGUGGAGUUCUCGGAAGAACCGUUCGGGGUGAUCGUCAGACGUCAACUUGAUGGUAGGGUAUUGCUGAACACAACGGUCGCCCCCUUGUUUUUCGCCGACCAGUUUCUGCAGCUUUCGACAUCGCUGCCGUCCCAGUAUAUCACAGGGCUCGCGGAGCAUCUUUCACCCCUCAUGCUGAGCACGAGCUGGACACGGAUUACGCUCUGGAACAGGGAUCUCGCGCCGACGCCCGGAGCGAAUUUGUAUGGGUCGCAUCCCUUCUACCUCGCAUUGGAAGACGGGGGUUCCGCGCACGGAGUAUUCCUGCUUAAUUCUAAUGCGAUGGACGUUGUCUUGCAGCCCUCCCCUGCUUUGUCGUGGCGUUCCACGGGGGGCAUUUUGGACGUUUACAUCUUUUUGGGACCCGAGCCAAAGAGCGUAGUGCAGCAGUAUUUGGAUGUAGUGGGCUACCCCUUCAUGCCGCCUUAUUGGGGACUGGGGUUCCAUCUCUGCCGCUGGGGGUACUCUUCGACCGCGAUCACCCGCCAGGUGGUCGAGAACAUGACCAGAGCACAUUUCCCUUUGGACGUGCAGUGGAAUGAUUUGGAUUACAUGGAUAGCCGAAGAGACUUCACGUUCAAUAAGGACGGGUUUAGAGAUUUUCCCGCGAUGGUGCAAGAAUUGCACCAGGGUGGGCGCAGAUACAUGAUGAUCGUCGAUCCCGCCAUCAGCAGCUCGGGACCAGCGGGGAGUUACCGGCCUUACGAUGAGGGACUUAGGAGAGGCGUCUUUAUCACGAACGAAACAGGUCAGCCGCUCAUUGGUAAAGUGUGGCCUGGAUCAACGGCCUUUCCCGACUUCACGAAUCCCACAGCCCUCGCCUGGUGGGAAGACAUGGUGGCGGAGUUUCACGACCAAGUACCGUUUGAUGGGAUGUGGAUUGAUAUGAACGAACCCUCAAACUUUAUUCGCGGCUCGGAAGAUGGAUGCCCGAAUAAUGAGCUUGAGAAUCCCCCGUAUGUGCCAGGGGUGGUAGGUGGGACGCUCCAGGCCGCUACGAUCUGUGCGUCAUCACAUCAGUUCUUGUCAACGCACUACAACUUGCACAAUCUUUACGGUUUGACUGAAGCCAUCGCUUCGCAUCGCGCGCUGGUCAAAGCGCGUGGUACGCGACCCUUCGUUAUUUCUCGGUCCACAUUUGCCGGGCACGGUCGGUAUGCCGGACACUGGACGGGAGAUGUCUGGUCUAGCUGGGAGCAGCUCGCGUCGAGCGUACCGGAGAUCCUCCAGUUCAAUCUUUUGGGAGUUCCGCUCGUCGGCGCUGACGUGUGCGGUUUUCUCGGAAACACAUCAGAAGAGCUUUGCGUACGCUGGACACAGCUCGGUGCGUUUUACCCCUUUAUGAGAAACCAUAACUCGUUGCUCUCACUCCCUCAAGAGCCGUACAGUUUUUCGGAGCCUGCGCAACAGGCGAUGCGGAAGGCAUUGACACUUCGCUAUGCACUGCUCCCGCAUCUCUAUACUCUGUUCCAUCAGGCCCAUGUGGCUGGAGAAACGGUGGCGAGGCCCCUGUUCUUGGAGUUCCCCAAAGAUAGUUCCACAUGGACCGUGGAUCACCAGUUGCUGUGGGGAGAGGCGCUUCUGAUCACUCCGGUACUUCAGGCGGGUAAAGCGGAAGUCACUGGGUAUUUCCCGCUUGGGACCUGGUACGACCUUCAGACUGUCCCAGUAGAAGCCCUCGGAAGCCUGCCACCUCCCCCUGCUGCACCCCGCGAGCCUGCAAUCCAUAGCGAGGGCCAGUGGGUAACGUUGCCAGCCCCACUGGAUACCAUCAAUGUCCACCUCAGGGCGGGUUACAUUAUCCCUCUCCAAGGCCCUGGGUUGACCACCACAGAGUCGCGCCAGCAGCCAAUGGCACUUGCGGUCGCAUUGACGAAAGGGGGUGAAGCCCGAGGGGAACUGUUUUGGGAUGACGGGGAAAGCCUUGAGGUGCUGGAACGGGGAGCGUACACACAAGUCAUUUUCUUGGCCAGGAACAACACUAUUGUCAACGAGUUGGUGCGCGUGACCUCUGAGGGUGCCGGACUGCAACUGCAGAAGGUCACGGUCCUCGGAGUGGCGACAGCACCCCAACAGGUCCUUAGUAACGGAGUACCUGUCUCGAACUUUACAUACUCCCCGGACACGAAGGUGCUCGACAUCUGUGUGUCGCUGCUUAUGGGGGAACAGUUUCUCGUGAGCUGGUGCUAGCGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAA UUAAGUUGCAUCAAGCU.

In another example, a full length codon-optimized human acidalpha-glucosidase (GAA) messenger RNA sequence is shown below:

[SEQ ID NO: 12] GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGGGAGUCAGACACCCGCCGUGCUCGCACAGGCUUCUGGCCGUGUGCGCACUCGUGAGUCUGGCGACUGCUGCGUUGCUGGGGCACAUUCUUCUCCACGACUUUCUCUUGGUGCCCCGAGAAUUGUCGGGCUCGUCGCCGGUACUGGAAGAAACCCACCCCGCACAUCAGCAGGGCGCGUCGCGGCCUGGUCCGAGGGAUGCCCAGGCACAUCCCGGAAGGCCACGAGCCGUCCCGACUCAAUGUGACGUACCUCCCAAUUCCCGGUUCGACUGUGCGCCAGACAAGGCAAUCACGCAAGAGCAGUGCGAAGCCCGUGGAUGCUGCUAUAUUCCGGCGAAGCAGGGACUUCAGGGAGCCCAGAUGGGGCAGCCCUGGUGUUUCUUCCCGCCUUCCUAUCCCUCAUAUAAGCUGGAGAAUUUGUCGUCCUCGGAAAUGGGGUAUACCGCUACUCUUACGAGAACCACCCCCACAUUCUUUCCGAAGGACAUCCUUACUCUGCGGCUCGACGUGAUGAUGGAGACAGAAAAUAGGCUGCAUUUCACGAUCAAAGACCCGGCGAACCGGAGAUAUGAGGUUCCGCUUGAGACUCCCCACGUUCACUCUCGUGCGCCUUCACCCUUGUACUCCGUGGAGUUCUCGGAAGAACCGUUCGGGGUGAUCGUCAGACGUCAACUUGAUGGUAGGGUAUUGCUGAACACAACGGUCGCCCCCUUGUUUUUCGCCGACCAGUUUCUGCAGCUUUCGACAUCGCUGCCGUCCCAGUAUAUCACAGGGCUCGCGGAGCAUCUUUCACCCCUCAUGCUGAGCACGAGCUGGACACGGAUUACGCUCUGGAACAGGGAUCUCGCGCCGACGCCCGGAGCGAAUUUGUAUGGGUCGCAUCCCUUCUACCUCGCAUUGGAAGACGGGGGUUCCGCGCACGGAGUAUUCCUGCUUAAUUCUAAUGCGAUGGACGUUGUCUUGCAGCCCUCCCCUGCUUUGUCGUGGCGUUCCACGGGGGGCAUUUUGGACGUUUACAUCUUUUUGGGACCCGAGCCAAAGAGCGUAGUGCAGCAGUAUUUGGAUGUAGUGGGCUACCCCUUCAUGCCGCCUUAUUGGGGACUGGGGUUCCAUCUCUGCCGCUGGGGGUACUCUUCGACCGCGAUCACCCGCCAGGUGGUCGAGAACAUGACCAGAGCACAUUUCCCUUUGGACGUGCAGUGGAAUGAUUUGGAUUACAUGGAUAGCCGAAGAGACUUCACGUUCAAUAAGGACGGGUUUAGAGAUUUUCCCGCGAUGGUGCAAGAAUUGCACCAGGGUGGGCGCAGAUACAUGAUGAUCGUCGAUCCCGCCAUCAGCAGCUCGGGACCAGCGGGGAGUUACCGGCCUUACGAUGAGGGACUUAGGAGAGGCGUCUUUAUCACGAACGAAACAGGUCAGCCGCUCAUUGGUAAAGUGUGGCCUGGAUCAACGGCCUUUCCCGACUUCACGAAUCCCACAGCCCUCGCCUGGUGGGAAGACAUGGUGGCGGAGUUUCACGACCAAGUACCGUUUGAUGGGAUGUGGAUUGAUAUGAACGAACCCUCAAACUUUAUUCGCGGCUCGGAAGAUGGAUGCCCGAAUAAUGAGCUUGAGAAUCCCCCGUAUGUGCCAGGGGUGGUAGGUGGGACGCUCCAGGCCGCUACGAUCUGUGCGUCAUCACAUCAGUUCUUGUCAACGCACUACAACUUGCACAAUCUUUACGGUUUGACUGAAGCCAUCGCUUCGCAUCGCGCGCUGGUCAAAGCGCGUGGUACGCGACCCUUCGUUAUUUCUCGGUCCACAUUUGCCGGGCACGGUCGGUAUGCCGGACACUGGACGGGAGAUGUCUGGUCUAGCUGGGAGCAGCUCGCGUCGAGCGUACCGGAGAUCCUCCAGUUCAAUCUUUUGGGAGUUCCGCUCGUCGGCGCUGACGUGUGCGGUUUUCUCGGAAACACAUCAGAAGAGCUUUGCGUACGCUGGACACAGCUCGGUGCGUUUUACCCCUUUAUGAGAAACCAUAACUCGUUGCUCUCACUCCCUCAAGAGCCGUACAGUUUUUCGGAGCCUGCGCAACAGGCGAUGCGGAAGGCAUUGACACUUCGCUAUGCACUGCUCCCGCAUCUCUAUACUCUGUUCCAUCAGGCCCAUGUGGCUGGAGAAACGGUGGCGAGGCCCCUGUUCUUGGAGUUCCCCAAAGAUAGUUCCACAUGGACCGUGGAUCACCAGUUGCUGUGGGGAGAGGCGCUUCUGAUCACUCCGGUACUUCAGGCGGGUAAAGCGGAAGUCACUGGGUAUUUCCCGCUUGGGACCUGGUACGACCUUCAGACUGUCCCAGUAGAAGCCCUCGGAAGCCUGCCACCUCCCCCUGCUGCACCCCGCGAGCCUGCAAUCCAUAGCGAGGGCCAGUGGGUAACGUUGCCAGCCCCACUGGAUACCAUCAAUGUCCACCUCAGGGCGGGUUACAUUAUCCCUCUCCAAGGCCCUGGGUUGACCACCACAGAGUCGCGCCAGCAGCCAAUGGCACUUGCGGUCGCAUUGACGAAAGGGGGUGAAGCCCGAGGGGAACUGUUUUGGGAUGACGGGGAAAGCCUUGAGGUGCUGGAACGGGGAGCGUACACACAAGUCAUUUUCUUGGCCAGGAACAACACUAUUGUCAACGAGUUGGUGCGCGUGACCUCUGAGGGUGCCGGACUGCAACUGCAGAAGGUCACGGUCCUCGGAGUGGCGACAGCACCCCAACAGGUCCUUAGUAACGGAGUACCUGUCUCGAACUUUACAUACUCCCCGGACACGAAGGUGCUCGACAUCUGUGUGUCGCUGCUUAUGGGGGAACAGUUUCUCGUGAGCUGGUGCUAGGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAU UAAGUUGCAUCAAAGCU.Exemplary Formulation Protocols

A. cKK-E12

Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, cholesteroland DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA was prepared from a 1 mg/mL stock. The lipid solutionwas injected rapidly into the aqueous mRNA solution and shaken to yielda final suspension in 20% ethanol. The resulting nanoparticle suspensionwas filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. Final concentration=0.64 mg/mL GAA mRNA (encapsulated).Z_(ave)=80 nm; PDI=0.17. % Encapsulation=85%; Yield=89%.

B. C12-200

Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, cholesteroland DMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

C. HGT4003

Aliquots of 50 mg/mL ethanolic solutions of HGT4003, DOPE, cholesteroland DMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

D. ICE

Aliquots of 50 mg/mL ethanolic solutions of ICE, DOPE, cholesterol andDMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

E. HGT5001

Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesteroland DMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

F. HGT5000

Aliquots of 50 mg/mL ethanolic solutions of HGT5000, DOPE, cholesteroland DMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

G. DLinKC2DMA

Aliquots of 50 mg/mL ethanolic solutions of DLinKC2DMA, DOPE,cholesterol and DMG-PEG2K are mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of GAA mRNA is prepared from a 1 mg/mLstock. The lipid solution is injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension is filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. The final concentration,Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of the GAA encapsulated mRNA are determined.

H. DODAP

Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE, cholesterol andDMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

I. DODMA

Aliquots of 50 mg/mL ethanolic solutions of DODMA, DOPE, cholesterol andDMG-PEG2K are mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GAA mRNA is prepared from a 1 mg/mL stock. The lipid solution isinjected rapidly into the aqueous mRNA solution and shaken to yield afinal suspension in 20% ethanol. The resulting nanoparticle suspensionis filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. The final concentration, Z_(ave), Dv₍₅₀₎ and Dv₍₉₀₎ of theGAA encapsulated mRNA are determined.

Example 2. Intravenous Administration of GAA mRNA-Loaded LiposomeNanoparticles

This example illustrates exemplary methods of administering GAAmRNA-loaded liposome nanoparticles and methods for analyzing GAA mRNAand glycogen in various target tissues in vivo.

All studies were performed using GAA knock out mice. Mice were treatedwith human GAA mRNA-loaded cKK-E12-based lipid nanoparticles by a singlebolus tail-vein injection of a 1.0 mg/kg dose. Mice were sacrificed andperfused with saline at 30 minutes, 3 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours and 7 days.

Tissues, such as liver and muscle, of each mouse were harvested,apportioned into separate parts, and stored in either 10% neutralbuffered formalin or snap-frozen and stored at −80° C. for analysis.

Direct detection of the active pharmaceutical ingredient (GAA mRNA) inthe muscle of the treated mice was achieved using in situ hybridization(ISH) based methods. As demonstrated in FIGS. 1A and 1B, the exogenoushuman GAA messenger RNA was detected at 6 hours and 12 hours.

Liver glycogen levels were reduced following administration of the GAAmRNA lipid nanoparticle (FIG. 2A) as compared to liver glycogen levelsin untreated GAA knock out mice (FIG. 2B).

Example 3. Intramuscular Administration of GAA mRNA-Loaded LiposomeNanoparticles

This example illustrates exemplary methods of administering GAAmRNA-loaded liposome nanoparticles and methods for analyzing GAA mRNAand glycogen in various target tissues in vivo.

All studies were performed using GAA knock out mice. Mice were treatedwith human GAA mRNA-loaded cKK-E12-based lipid nanoparticles by a singleintramuscular injection of a 1.0 mg/kg dose. Mice were sacrificed andperfused with saline at 24 hours.

Tissues, such as liver and muscle, of each mouse were harvested,apportioned into separate parts, and stored in either 10% neutralbuffered formalin or snap-frozen and stored at −80° C. for analysis.

Direct detection of the active pharmaceutical ingredient (GAA mRNA) inthe muscle of the treated mice was achieved using in situ hybridization(ISH) based methods. As demonstrated in FIG. 3 , the exogenous human GAAmessenger RNA was detected at high levels at 24 hours.

Quadriceps muscle glycogen levels were reduced following administrationof the GAA mRNA lipid nanoparticle (FIGS. 4A and 4B) as compared toquadriceps muscle glycogen levels in untreated GAA knock out mice (FIGS.4C and 4D).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims.

We claim:
 1. A method of treating Pompe disease, comprisingadministering to a subject in need of treatment a composition comprisingan mRNA encoding acid alpha-glucosidase (GAA) at an effective dose andan administration interval such that at least one symptom or feature ofPompe disease is reduced in intensity, severity, or frequency or hasdelayed onset, wherein the mRNA is at least 90% identical to SEQ ID NO:3.
 2. The method of claim 1, wherein administering the compositionresults in treatment of hypertrophic cardiomyopathy in the subject. 3.The method of claim 1, wherein the mRNA is encapsulated within aliposome.
 4. The method of claim 3, wherein the liposome comprises oneor more cationic lipids, one or more non-cationic lipids, one or morecholesterol-based lipids and one or more PEG-modified lipids.
 5. Themethod of claim 4, wherein the one or more PEG-modified lipids comprisea poly(ethylene) glycol chain of up to 5 kDa in length covalentlyattached to a lipid with alkyl chain(s) of C₆-C₂₀ length.
 6. The methodof claim 4, wherein the cationic lipid constitutes about 30-50% of theliposome by weight.
 7. The method of claim 1, wherein the mRNA isadministered at the effective dose ranging from about 0.1-5.0 mg/kg bodyweight.
 8. The method of claim 1, wherein the composition isadministered intravenously.
 9. The method of claim 1, wherein thecomposition is administered intramuscularly.
 10. The method of claim 1wherein the administering of the composition results in GAA proteinexpression in liver.
 11. The method of claim 1, wherein theadministering of the composition results in GAA protein expression inserum.
 12. The method of claim 1, wherein the mRNA is codon optimized.13. The method of claim 1, wherein the mRNA comprises one or moremodified nucleotides.
 14. The method of claim 1, wherein the mRNA isunmodified.
 15. A composition for treating Pompe disease, comprising anmRNA encoding acid alpha-glucosidase (GAA) at an effective dose amountencapsulated within a liposome, wherein the mRNA is at least 90%identical to SEQ ID NO: 3, and wherein the liposome comprises a cationiclipid cKK-E12:


16. The composition of claim 15, wherein the liposome further comprisesone or more non-cationic lipids, one or more cholesterol-based lipids,and one or more PEG-modified lipids.
 17. The composition of claim 15,wherein the cationic lipid constitutes about 30-50% of the liposome bymolar ratio.
 18. The composition of claim 15, wherein the composition isformulated for intravenous administration.
 19. A composition fortreating Pompe disease, comprising an mRNA encoding acidalpha-glucosidase (GAA) at an effective dose amount encapsulated withina liposome, wherein the mRNA is at least 90% identical to SEQ ID NO: 3,and further wherein the liposome comprises cationic or non-cationiclipid, cholesterol-based lipid and PEG-modified lipid.
 20. Thecomposition of claim 19, wherein the mRNA further comprises SEQ ID NO: 8and SEQ ID NO: 9, or SEQ ID NO: 10.